Compositions and methods for inhibiting tumor cells by inhibiting the transcription factor atf5

ABSTRACT

The present invention relates to methods for treating and/or preventing tumors and/or promoting apoptosis in a neoplastic cell comprising contacting the neoplastic cell with an cell-penetrating dominant-negative ATF5 (“CP-d/n-ATF5”), wherein the CP-d/n-ATF5 is capable of inhibiting ATF5 function and/or activity.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/830,194, filed Aug. 19, 2015, which is a continuation ofInternational Patent Application No. PCT/US2014/017550, filed Feb. 21,2014, and claims priority to U.S. Provisional Application Ser. No.61/768,390, filed Feb. 22, 2013, to all of which priority is claimed andthe contents of which are incorporated herein in their entireties.

STATEMENT OF GOVERNMENT INTEREST

This invention was made with government support under Grant No.RCA126924A awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jan. 13, 2017, is22,000 bytes in size.

BACKGROUND OF THE INVENTION

Approximately one million people are diagnosed with cancer each year,and many millions of Americans of all ages are currently living withsome form of cancer. At some time during the course of their lifetime,one out of every two American men and one out of every three Americanwomen will be diagnosed with some form of cancer. Of the one millionAmericans diagnosed with cancer annually, 17,000 are diagnosed withbrain tumors. Brain tumors invade and destroy normal tissue, producingsuch effects as impaired sensorimotor and cognitive function, increasedintracranial pressure, cerebral edema, and compression of brain tissue,cranial nerves, and cerebral vessels. Drowsiness, lethargy, obtuseness,personality changes, disordered conduct, and impaired mental facultiesare the initial symptoms in 25% of patients with malignant brain tumors.Treatment of brain tumors is often multimodal, and depends on pathologyand location of the tumors. For malignant gliomas, multimodal therapy,including chemotherapy, radiation therapy, and surgery, is used to tryto reduce tumor mass. Regardless of approach, however, prognosis forpatients suffering from these tumors is guarded: the median term ofsurvival after chemotherapy, radiation therapy, and surgery is onlyabout 1 year, and only 25% of these patients survive for 2 years.

The prevalence of cancer, and in particular brain tumors refractory toexisting therapies, has led to the identification of transcriptionfactors impacting cell cycle control of neuronal cells, including ATF5(Acharay et al., J Struct Biol 155:130-139 (2006)). ATF5 belongs to theactivating transcription factor/CREB family of basic leucine zippertranscription factors (Acharay et al., J Struct Biol 155:130-139 (2006);Greene et al., J Neurochem 108:11-22 (2009)). ATF5 is highly expressedby neural stem and progenitor cells for neuronal and glial lineages andits expression plummets when these differentiate (Angelastro et al., JNeurosci 23:4590-4600 (2003); Angelastro et al., J Neurosci 25:3889-3899(2005); Mason et al., Mol Cell Neurosci 29:372-380 (2005)). Becauseconstitutive ATF5 expression in neural progenitor cells causes them toremain in cell cycle and blocks their differentiation, (Angelastro etal., J Neurosci 23:4590-4600 (2003); Angelastro et al., J Neurosci25:3889-3899 (2005); Mason et al., Mol Cell Neurosci 29:372-380 (2005)),ATF5 expression in GBM was assayed as GBMs are thought to be derivedfrom neural stem and progenitor cells (Tanaka et al., Nat Rev Clin Oncol10:14-26 (2012)). Examination of 29 resected GBMs revealed high ATF5expression by all and by all 9 rodent and human GBM lines examined(Angelastro et al., Oncogene 25:907-916 (2006)). These findings havebeen corroborated and additional data has indicated a correlationbetween ATF5 levels and GBM prognosis (Dong et al., J Neuropathol ExpNeurol 64:948-955 (2005); Sheng et al., Nat Med 16:671-677 (2010)).

To examine the role of ATF5 in GBM, a dominant-negative inhibitor of theprotein was created to interfere with ATF5 function (Acharay et al., JStruct Biol 155:130-139 (2006), Angelastro et al., Oncogene 25:907-916(2006)), and si/shRNAs were developed to silence its expression. Cultureexperiments with human and rat GBM lines showed that both the d/n-ATF5and the ATF5 si/shRNAs cause their massive apoptotic death (Angelastroet al., Oncogene 25:907-916 (2006)). In contrast, ATF5+ proliferatingneural progenitor cells and astrocytes did not show this apoptoticresponse. In an initial in vivo study, it was found that if the d/n-ATF5was retrovirally-delivered it would selectively and with very highefficiency kill tumor cells generated from implanted C6 rat GBM cells,but not normal proliferating brain cells (Angelastro et al., Oncogene25:907-916 (2006)). In subsequent studies, an adult mouse model was usedin which gliomas (of grades ranging form low-grade gliomas to GBMs) areefficiently generated by infection with a retrovirus expressing PDGF-Band a p53 shRNA (Arias et al., Oncogene 31:739-751 (2012)). Using miceengineered to conditionally express the d/n-ATF5 from the human GFAPpromoter (which is expressed in neural stem/progenitor cells, astrocytesand GBMs), induction of that d/n-ATF5 led to completeregression/eradication of tumors and survival of all 24 treated mice.Likewise, expression of the d/n-ATF5, prior to injection of thePDGF-B/shRNA-p53 retrovirus, prevented tumor development in 85.7% of themice. In contrast, for mice in which the d/n-ATF5 was not induced, 15/16had tumors and 40% died within the test period. There were no apparenteffects on normal cells (Arias et al., Oncogene 31:739-751 (2012)).

SUMMARY OF THE INVENTION

In certain embodiments, the present invention relates to methods fortreating and/or preventing tumors and/or promoting apoptosis in aneoplastic cell comprising contacting the neoplastic cell with ancell-penetrating dominant-negative ATF5 (“CP-d/n-ATF5”), wherein theCP-d/n-ATF5 is capable of inhibiting ATF5 function and/or activity.

In certain embodiments, the neoplastic cell is selected from the groupconsisting of: breast, ovary, endometrium, gastric, colon, liver,pancreas, kidney, bladder, prostate, testis, skin (e.g.,melanocyte/melanoma cell), esophagus, tongue, mouth, parotid, larynx,pharynx, lymph node, lung, blood (e.g., hematological cancers),peripheral nervous system, and brain. In certain embodiments, theneoplastic cell is selected from the group consisting of glioblastoma,astrocytoma, glioma, medulloblastoma, meningioma, mesothelioma, andneuroblastoma. In certain embodiments, the neoplastic cell is associatedwith a primary or a recurrent brain tumor.

In certain embodiments the CP-d/n-ATF5 is administered orally,parenterally (e.g., subcutaneously), intranasally, and/or transdermally.

In certain embodiments the CP-d/n-ATF5 comprises a portion of the human,rat, or mouse ATF5 peptide sequence or a combination thereof. In certainembodiments, the cell-penetrating dominant-negative ATF5 comprises asequence selected from the group consisting of:

(SEQ ID NO: 1) LEQENAE, (SEQ ID NO: 2) LEKEAEELEQENAE, (SEQ ID NO: 3)LARENEELLEKEAEELEQENAE, (SEQ ID NO: 4) LEQRAEELAREN EELLEKEAEELEQENAE,or (SEQ ID NO: 4) LEQRAEELARENEELLEKEAEELEQENAE,linked to a peptide that forms a leucine zipper and to acell-penetrating sequence. In certain embodiments, the CP-d/n-ATF5comprises a sequence selected from the group consisting of:

(SEQ ID NO: 5) LEQENAELEGECQGLEARNRELKERAES, (SEQ ID NO: 6)LEKEAEELEQENAELEGECQGLEARNRELK ERAES, (SEQ ID NO: 7)LARENEELLEKEAEELEQENAELEGECQGLEARNRELKERAES, (SEQ ID NO: 8)LEQRAEELAR NEELLEKEAEELEQENAELEGECQGLEARNRELKERA ES, (SEQ ID NO: 9)LEQRAEELARENEELLEKEAEELEQENAELEGECQGLEARNRELKERA ESV,where the underlined sequence is the dominant-negative sequence and theremainder of the sequence is the ATF5 leucine zipper, and the sequenceis operably linked to (in frame) a cell-penetrating sequence. In certainembodiments, the cell-penetrating dominant-negative ATF5 comprises asequence selected from the group consisting of:

(SEQ ID NO: 10) LEQENAELEGECQGLEARNRELRERAES, (SEQ ID NO: 11)LEKEAEELEQENAEL EGECQGLEARNRELRERAES, (SEQ ID NO: 12)LARENEELLEKEAEELEQENAELEGECQGLEARNREL RERAES, (SEQ ID NO: 13)LEQRAEELARENEELLEKEAEELEQENAELEGECQGLEARNRELRERA ES, (SEQ ID NO: 14)LEQRAEELARENEELLEKEAEELEQENAELEGECQGLEARNRELRERA ESV,where the underlined sequence is the dominant-negative sequence and theremainder of the sequence is the ATF5 leucine zipper, and the sequenceis operably lined to a cell-penetrating sequence.

In certain embodiments, the cell-penetrating dominant-negative ATF5comprises a sequence selected from the group consisting of: (1)

(SEQ ID NO: 15) MGSSHHHHHHSSGLVPRGSHM RQIKIWFQNRRMKWKK DYKDDDDKMASMTGGQQMGRDPD

LEGECQG LEARNRELRERAES V ,where the underlined residues (MG-HM) are a 6XHis-tag leader sequence(“6XHis” disclosed as SEQ ID NO: 16), the bold residues

are a Penetratin sequence, the italicized residues (DY-DK) are a Flagtag, the residues with no font modification (MA-PD) are spacer aminoacids, the bold and italicized residues

are a d/n sequence, and the bold and underlined residues

are an ATF5 leucine zipper truncated after its first Valine; (2)

(SEQ ID NO: 17) MGSSHHHHHHSSGLVPRGSHMLE YGRKKRRQRRR YPYDVPDYAMASMTGGQQMGRDPD

LEGECQG LEARNRELRERAESV ,where the underlined residues (MG-LE) are a 6XHis-tag leader sequence(“6XHis” disclosed as SEQ ID NO: 16), the bold residues

are a TAT sequence, the italicized residues (YP-YA) are an HA tag, theresidues with no font modification (MA-PD) are spacer amino acids, thebold and italicized residues

are a d/n sequence, and the bold and underlined residues

are an ATF5 leucine zipper truncated after its first Valine; (3)

(SEQ ID NO: 18) MGSSHHHHHHSSGLVPRGSHM RQIKIWFQNRRMKWKK LEQRAEELARENEELLEKEAEELEQENAE LEGECQGLEARNRELKERAESVwhere the where the underlined residues (MG-HM) are a 6XHis-tag leadersequence (“6XHis” disclosed as SEQ ID NO: 16), the bold residues

are a Penetratin sequence, the italicized residues (LE-AE) are a d/nsequence, and the bold and underlined residues

are an ATF5 leucine zipper truncated after its first Valine; and (4)

(SEQ ID NO: 19) RQIKIWFQNRRMKWKK LEQRAEELARENEELLEKEAEELEQENAE LEGECQGLEARNRELKERAESVwhere the bold residues

are a Penetratin sequence, the italicized residues (LE-AE) are a d/nsequence, and the bold and underlined residues

are an ATF5 leucine zipper truncated after its first Valine. In certainembodiments, the cell-penetrating dominant-negative ATF5 is chemicallysynthesized.

In certain embodiments, the invention also relates to kits for use intreating and/or preventing tumors and/or promoting apoptosis in aneoplastic cell. Additional aspects of the present invention will beapparent in view of the description which follows.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 GFP-d/n-ATF5 C-terminally truncated fusion protein(GFP-d/n-ATF5-Tr) promotes the same level of apoptosis as full-lengthGFP-d/n-ATF5 protein in C6 glioma cells. C6 cells were transfected withpQC-X-I-eGFP, pQC-d/n-GFPATF5, or pQC-GFPATF5-tr. The percentages(mean±SEM, n=4; total of approximately 200 cells scored per condition)of condensed apoptotic nuclei in GFP+ transfected cells were determined2 days later. Student's t-test; GFP+ cells versus GFP-d/n-ATF5+ cells orGFP-d/n-ATF5-tr cells, (*p<0.05); GFP-d/n-ATF5+ cells versusGFP-d/n-ATF5-tr cells, (Not Significant).

FIG. 2A-2C Purity and molecular properties of bacterially-expressed andpurified 6xhistidine-Flag-Tagged Penetratin-Flag-D/N-ATF5-tr(Pen-d/n-ATF5-RP) (“6xhistidine” disclosed as SEQ ID NO: 16) and6xhistidine-Flag-Tagged Penetratin-Flag-Control (Pen-control-RP)peptides (“6xhistidine” disclosed as SEQ ID NO: 16). (FIG. 2A) Coomassiestained SDS-PAGE of purified Pen-d/n-ATF5-RP and Pen-control-RP (5 μgper lane). Molecular weight markers are shown on the left, and a linearscheme of each peptide is shown above each lane. Purification was asdescribed in Methods. (FIG. 2B) Deconvoluted mass spectra from LC-highResolution mass spectrometry of purified Pen-d/n-ATF5-RP. The mostabundant species is the 12,948.88 Da monomer form withoutformyl-methionine followed by the formyl-methionine 13,127 Da monomerform (isoform). The spectrum also reveals a small amount of the 25,897.5Da dimer. (FIG. 2C) Stability of Pen-d/n-ATF5-RP in Human Serum.Pen-d/n-ATF5-RP (36 μM) was incubated with human serum (25% v/v in PBS)at 37° C. for 0 to 48 h. Aliquots were withdrawn at various times andthe Pen-d/n-ATF5-RP peptide was resolved by SDS-PAGE, transferred toPVDF membrane and probed with anti-Flag antibody. The anti-Flag signalwas detected by near IR using LiCor software and densitometry of theband at the expected size of Pen-d/n-ATF5-RP was performed andquantified using ImageJ. Values are mean±SEM, n=3.

FIG. 3A-3B Uptake and retention of Pen-d/n-ATF5-RP by culturedglioblastoma cells. (FIG. 3A) Confocal images of C6 rat glioblastomacells incubated for 4 hours with either 200 nM Pen-control-RP (left) orPen-d/n-ATF5-RP (right). Cells were washed, fixed and stained withanti-Flag (red) and DAPI (blue). Scale bar=2 μm. (FIG. 3B) Rat C6 andhuman U87 glioblastoma cells were incubated for the indicated times with3 μM Pen-d/n-ATF5-RP, washed, fixed and immunostained with anti-Flag(green) and DAPI (blue). Scale bar=5 μm.

FIG. 4 Pen-d/n-ATF5-RP promotes apoptosis of C6 glioblastoma cells. C6cells were treated with 3 μM Pen-d/n-ATF5-RP or 3 μM Pen-Control-RP, orwere untreated. The percentage (mean±SEM; n=4 in 2 independentexperiments; approximately 200 cells scored) of condensed apoptoticnuclei in cells was determined 5 days later. Student's t-test;Pen-d/n-ATF5-RP versus Pen-Control-RP cells or nontreated, (*p<0.05);Pen-Control-RP cells versus nontreated cells, (p=0.29).

FIG. 5A-5F Pen-d/n-ATF5-RP enters the mouse brain and causes targetedapoptosis of glioma cells. (FIG. 5A-5F) Representative brain sectionsstained with Flag antibody to indicate presence of Pen-d/n-ATF5-RP or HAto identify presence of tumor-inducing retrovirus (red); TUNEL toidentify apoptosis (green) and DAPI to localize nuclei (blue). (FIG. 5A)Murine brain tumor 24 h post-treatment (16 h after last injection) withPen-d/n-ATF5-RP (52 days post-retrovirus injection). (FIG. 5B) Normalcontralateral cerebral hemisphere of the same mouse in FIG. 5A. (FIG.5C) Murine brain tumor 24 h post-injection with saline (59 dayspost-retrovirus injection). Presence of Pen-d/n-ATF5 within cells isconfirmed in the treated mouse (FIG. 5A, 5B) versus saline control (FIG.5C) by increased Flag antibody staining. Glioma cell-specific inductionof apoptosis by Pen-d/n-ATF5-RP is illustrated by increased TUNELstaining (green) in FIG. 5A as compared to FIG. 5B and FIG. 5C. (FIG.5D) TUNEL and DAPI staining of a tumor-containing brain section 160 dayspost-retrovirus injection and 3 days after injection of Pen-control-RP.Note HA+ cells identifying tumor cells and absence of TUNEL staining.(FIG. 5E) Staining as in FIG. 5D of a tumor-containing section (143 dayspost-retrovirus injection) and 3 days after Pen-d/n-ATF5-RP treatment.Note the presence of TUNEL staining in HA+ tumor cells and fragmentedappearance of the staining as compared to FIG. 5A and FIG. 5D. (FIG. 5F)Staining as in FIG. 5D of a tumor-containing section 150 days afterretrovirus injection and 2 days after 2 treatments of subcutaneousPen-d/n-ATF5-tr-RP injections. Note the qualitative similarity ofstaining pattern to FIG. 5E with fragmented PDGF-B-HA and TUNELstaining. Scale bars equal 20 μm.

FIG. 6A-6D Retention of Pen-d/n-ATF5-RP in mouse brain at various timesafter administration. Mice received 4 intraperitoneal injections ofeither saline (FIG. 6A) or Pen-d/n-ATF5-RP (FIG. 6B, 6C) as described inthe text. Animals were sacrificed at either 40 (FIG. 6A, 6B) or 64 (FIG.6C) h after the last injection and sections of their fixed brains werestained with either anti-Flag (Red; to visualize Pen-d/n-ATF5-RP) orDAPI (blue; to visualize nuclei). (FIG. 6D) Densitometry of anti-FlagImmunostaining. The optical densities (red channel) of fifteen random0.176 inch2 areas were determined in each of the images and averaged±SDusing Image J. Student's t-test; Pen-d/n-ATF5-RP (64 hours) or (40hours) versus saline, (*p<0.05). Scale bar is 10 μm.

FIG. 7A-7D′ H&E staining of the SVZ and hippocampal dentate gyms showsno detectable difference between these structures inPen-d/n-ATF5-RP-treated and non-treated mice. (FIG. 7A, 7A′) Lateralventricle/SVZ (FIG. 7A) and hippocampal dentate gyms (FIG. 7A′) from atumor-bearing mouse 183 days after the second set of subcutaneoustreatments with Pen-d/n-ATF5-RP. (FIG. 7B, 7B′) Lateral ventricle/SVZ(FIG. 7B) and hippocampal dentate gyms (FIG. 7B′) from an age-matchedcontrol mouse not treated with Pen-d/n-ATF5-RP and not injected withretrovirus. (FIG. 7C, 7C′) Lateral ventricle/SVZ (FIG. 7C) andhippocampal dentate gyms (FIG. 7C′) from a non-tumor-bearing mouse 1 dayafter the second set (given 5 days after the first set) of subcutaneoustreatments with Pen-d/n-ATF5-RP. (FIG. 7D, 7D′) Lateral ventricle/SVZ(FIG. 7D) and hippocampal dentate gyms (FIG. 7D′) from an age-matcheduntreated non-tumor-bearing control mouse. Scale for FIG. 7A-7B′ is 20μm and 50 μm for FIG. 7C-7D′.

FIG. 8A-8F Example of MRI and histopathology of a mouse glioma treatedwith Pen-Control-RP peptide. (FIG. 8A) Post-contrast 3D FLASH MRIcoronal image of the cerebrum of a control mouse that was not injectedwith PDGF-B-HA/sh-p53 retrovirus. (FIG. 8B) Post-contrast 3D FLASH MRIcoronal image of mouse cerebrum showing a bilateral tumor (whitecontrast) 246 days after PDGF-B-HA/shp53 retrovirus injection and priorto treatment with Pen-Control-RP peptide. (FIG. 8C) Post-contrast 3DFLASH MRI image of the same mouse brain 40 days after subcutaneoustreatment with Pen-Control-RP peptide (as described in the text) revealspersistence of the tumor (arrows). (FIG. 8D) H&E stained sections of thesame mouse brain at tumor-containing areas 1 and 2 shown by arrows inpanel FIG. 8C. The mouse was sacrificed 116 days after the secondtreatment with Pen-Control-RP peptide due to moribund behavior. Presenceof tumor is indicated in both sections by hyperchromatic nuclei andhigher cellularity. (FIG. 8E) Immunostaining for HA tag in sections fromareas 1 and 2 shown in FIG. 8C reveals presence of virally-deliveredPDGF-B-HA in induced tumor cells. (FIG. 8F) Immunostaining of sectionsfrom areas 1 and 2 shown in FIG. 8C reveals a high index ofKi67+/dividing cells indicative of tumor. Scale bars in FIG. 8D-8F are20 μm.

FIG. 9A-9E Pen-d/n-ATF5-RP promotes rapid and long-termregression/eradication of mouse glioma as indicated by MRI andhistology. (FIG. 9A) Post-contrast 3D FLASH MRI scans of a mouse brainbefore and at various times after treatment (as described in text) withPen-d/n-ATF5-RP. Pretreatment shows image of cortex 243 days afterPDGF-B-HA/shp53 retrovirus injection. Yellow arrows indicate location ofthe bilateral tumor. Post-treatment images of the same position of themouse cortex are at the indicated times after the second administrationof Pen-d/n-ATF5-RP. Yellow arrows in post-treatment images show locationof original tumor. (FIG. 9B) H&E image of the same mouse brain harvested192 days after the second Pen-d/n-ATF5-RP treatment. Region 1 representsthe location of the section as shown in the final time point in FIG. 9Aand at which the tumor was present before treatment. Note the absence ofhyperchromatic nuclei and higher cellularity that characterize gliomas.(FIG. 9C) Ki67 staining in region 2 (from Panel A/176 dayspost-treatment). Note the absence of Ki67+/proliferating cells seen ingliomas. (FIG. 9D) HA/DAPI staining of section from region 1. Note theabsence of cells expressing exogenous PDGF-B-HA. (FIG. 9E) GFAP/DAPIstaining of section region 1. Note clusters of GFAP+ cells consistentwith the presence of a glial scar where the tumor was formerly present.Lack of HA staining of a nearby section confirmed the absence of tumorcells. Diagonal green stripes are due to tissue folds. Scale bar is 20μm.

FIG. 10 MRI and histopathological images of an untreated mouse with abilateral tumor. Middle panel shows post-contrast 3D FLASH MRI image ofa tumor-bearing mouse brain at 112 days after PDGF-B-HA/shp53 retrovirusinjection. Panels (A) and (B) show images for sections stained with HAto reveal tumor cells and with DAPI to show nuclei. The yellow arrows onthe MRI along with the letters show the relative locations of the HA+sections shown in (A) and (B). Retroviral injection was on side B. Scalebar is 20 μm. DAPI (40,6-diamidino-2-phenylindole).

FIG. 11A-11E Second example illustrating that Pen-d/n-ATF5-RP promotesrapid and long-term regression/eradication of a mouse glioma asindicated by MRI and histology. (FIG. 11A) Post-contrast 3D FLASH MRIimages of a tumor-bearing mouse brain before and at various times aftertreatment with Pen-d/n-ATF5-RP. Pretreatment coronal and transverseimages (74 days after PDGF-B-HA/shp53 retrovirus injection) showmultifocal tumors within the cortex (arrows). Images from the same mousebrain are shown at 8, 21 and 181 days after two sets of subcutaneoustreatments with Pen-d/n-ATF5-RP as described in the text. Note decreasedsignal at 8 days and absence of detectable signals at 21 and 181 daysfollowing treatment. (FIG. 11A′, 11A″) Estimates of tumor volumecorroborate loss of signal by 8 days after Pen-d/n-ATF5-RP treatment.The same images as in FIG. 11A for pretreatment and 8 dayspost-treatment with arrows pointing to tumor foci (yellow circles) forwhich volumetric measurements were obtained using the region of interestelliptic cylinder tool (yellow circles). At pretreatment, the calculatedvolumes in FIG. 11A′ are 0.597 mm3, 0.164 mm3, and 0.760 mm3 for foci 1,2, and 3, respectively. For 8 days post-treatment (FIG. 11A″), volumesof the same tumors decreased to 0.106 mm3, 0.0302 mm3, and 0.0895 mm3for foci 1, 2, and 3, respectively. After 21 days of treatment thetumors could not be visualized for measurement. (FIG. 11B) H&E stainingof the same sacrificed mouse brain (183 days after treatment; 190 daysafter initial tumor detection) corroborates the absence of detectabletumor with the arrow pointing to the remnant scar corresponding to tumorfocus 1 shown in FIG. 11A′ and corroborates absence of detectable tumor.(FIG. 11C) HA immunostaining of the same brain (for PDGF-B-HA) indicatesthe absence of detectable tumor cells in the same focus 1 region as inFIG. 11A′ and FIG. 11B. (FIG. 11D) GFAP immunostaining of the same brainat focus 1 shows a remnant GFAP+ glial scar. (FIG. 11E) Ki67immunostaining of the focus 1 region of the same brain reveals theabsence of dividing cells. Scale bar is 20 μm for FIG. 11B-11E.

FIG. 12A-12C Long-term survival and tumor presence outcomes forglioma-bearing mice treated with Pen-d/n-ATF5-RP. (FIG. 12A) Survival ofglioma-bearing mice (verified by MRI) with or without treatment withPen-d/n-ATF5-RP (subcutaneous delivery as described in the text). Of thenine control mice, four control mice were treated with Pen-Control-RPpeptide and five were untreated. The experimental endpoint was 200 daysafter initial tumor detection by MRI. Survival analysis achieved bylog-rank test showed a p-value=0.0007(http://in-silico.net/tools/statistics/survivor). (FIG. 12B) MRIoutcomes for tumor-bearing mice before and after subcutaneous treatmentwith Pen-d/n-ATF5-RP as described in the text. The latter times rangefrom 176-225 days after tumor treatment (183-230 days after tumordetection). (FIG. 12C) Brain histopathological outcomes for tumors incontrol and Pen-d/n-ATF5-RP treated mice. In all cases, MRI verified thepresence of tumors prior to treatment. Control animals were as describedin FIG. 12A and brains were harvested either after death (6 controls),after the 6 month experimental endpoint (4 treated animals) or aftersacrifice for non-tumor related health problems (2 treated animals). Fortreated animals, histological analysis was carried out 260-438 daysafter tumor initiation (183-259 days after Pen-d/n-ATF5-RPadministration and 190-305 days after initial tumor detection). Sectionsof brain were prepared as described in Methods and were stained with H&Eand immunostained for Ki67 and HA (to identify PDGF-B-HA+ tumor cells).The presence/absence of tumors was based on observations ofhyperchromatic nuclei, high cellularity, elevated Ki67 staining and HAimmunostaining.

FIG. 13 indicates that TAT-d/n-ATF5 (TAT-ZIP) promotes apoptotic deathof cultured melanoma MEL501 cells. TAT-linked dominant-negative ATF5peptide at the indicated concentrations (in μM) was added to medium ofMEL501 melanoma cells. Four days later the cells were stained withHoescht dye and the cells were stained for proportion with apoptoticnuclei.

FIG. 14 indicates that TAT-d/n-ATF5 (TAT-ZIP) reduces the expression ofendogenous ATF5 in cultured U373 glioblastoma cells. TAT-linkeddominant-negative ATF5 peptide at the indicated concentrations (in μM)was added to medium of U373 glioblastoma cells for 17 hrs day and thecells were then harvested and analyzed by Western immunoblotting forlevels of endogenous ATF5. Note that the TAT-d/n-ATF5 greatly reducesexpression of endogenous ATF5. As previous studies have shown that tumorcells require endogenous ATF5 to survive, the mechanism of action bywhich the cell-penetrating TAT-ZIP peptide kills may be by causing lossof the endogenous ATF5 protein. Note also the smear above the endogenousATF5 when the TAT-ZIP peptide is present. This suggests that TAT-ZIPreduces endogenous ATF5 by causing its ubiquitination and proteasomaldegradation.

FIG. 15 indicates that TAT-d/n-ATF5 (TAT-ZIP) peptide induces expressionof the pro-death gene DDIT3 (CHOP) in various tumor cell lines. Cellswere treated with TAT-d/n-ATF5 for the indicated times at the indicateddoses (in μM) and then harvested and analyzed by Western immunoblottingfor expression of CHOP and other non-responsive proteins. Note theelevation of CHOP in all cases. Since CHOP may promote cell death, thesedata indicate that induction of CHOP protein may be one mechanism bywhich TAT-d/n-ATF5 kills tumor cells.

FIG. 16 indicates that silencing of CHOP protein with siRNA (top Westernimmunoblot) partially protects U87 cells from death caused byTAT-d/n-ATF5 peptide. Cells were treated with siCHOP to silence CHOPexpression (top Western immunoblot) or with control siRNA. They werethen exposed to TAT-d/n-ATF5 for 2 days and assessed for proportion ofcells with apoptotic nuclei. The data support the idea that part of themechanism by which TAT-d/n-ATF5 kills tumor cells is by increasing theirexpression of CHOP which in turn mediates death.

FIG. 17 indicates that TAT-D/N-ATF5 down-regulates BCL2 survivalprotein. Cultured U87 human glioblastoma cells were treated with theindicated concentrations of TATZIP (TAT-d/n-ATF5 peptide) (in μM) for 30hrs. The cells were then harvested and assessed by Westernimmunoblotting for expression of the survival protein BCL2. Thesefindings indicate that in addition to elevating pro-death CHOP,TAT-d/n-ATF5 may also kill tumor cells by reducing their levels of theBCL2 survival protein.

FIG. 18 indicates that TAT-D/N-ATF5 synergizes with temozolomide (TMZ)to kill cultured U87 glioblastoma cells. Cells were cultured for one daywith sub-lethal levels of TAT-d/n-ATF5 (TZIP 1 μM) and TMZ (50 μM)either separately or in combination, and then assessed for proportion ofcells with apoptotic nuclei. TMZ is presently the first-line treatmentfor human GBM. The data reveal that TAT-d/n-ATF5 not only functions inpresence of TMZ, but that the two drugs act in synergy to kill GBMcells. This suggests that TAT-d/n-ATF5 can be administered to patientswho are taking TMZ.

FIG. 19 depicts recombinant TAT-d/n-ATF5 (3 μM) treatment for 3-5 daysdecreasing viability of two human and one mouse GMB cell line asestablished by MTA assay.

FIG. 20 depicts synthetic PEN-d/n-ATF5 treatment decreasing cellviability of cultured U87 human glioblastoma cells as established by MTAassay. 5 days treatment at indicated concentrations (μM).

FIG. 21 depicts recombinant TAT-d/n-ATF5 treatment promoting death ofcultured U87 human glioblastoma cells as indicated by Annexin V/PIstaining and flow cytometry. Proportions of viable cells are shown inlower left quadrant (88% control vs 58% treated). Dying cell proportionsare in the lower right and upper right quadrants (9% in controls vs 36%in treated).

FIG. 22 depicts synthetic PEN-d/n-ATF5 promoting apoptotic death ofprimary GS9-6 human glioblastoma stem cells growing in culture asspheres. 6 days of treatment and data determined by Annexin V/PIstaining and flow cytometry

FIG. 23 depicts recombinant PEN-d/n-ATF5 promoting apoptotic death ofprimary GS9-6 human glioblastoma stem cells growing in culture asspheres. 5 days of treatment and data determined by Annexin V/PIstaining and flow cytometry.

DETAILED DESCRIPTION OF THE INVENTION ATF5 & D/N-ATF5 Compositions

ATF5 is widely expressed by various tumor types. In particular, ATF5 isexpressed not only in highly proliferative neural tumors, e.g.,glioblastomas, but is also expressed in multiple neoplasias including,but not necessarily limited to: breast, ovary, endometrium, gastric,colon, liver, pancreas, kidney, bladder, prostate, testis, skin,esophagus, tongue, mouth, parotid, larynx, pharynx, lymph node, lung,hematological cancers, peripheral nervous system, and brain tumors.

As used herein, “ATF5” includes both an “ATF5 protein” and an “ATF5analogue”. Unless otherwise indicated, “protein” shall include aprotein, protein domain, polypeptide, or peptide, and any fragmentthereof. The ATF5 protein has the amino acid sequence set forth in NCBIAccession No. NP_001180575 (human ATF5) or NCBI Accession No. NP_109618(murine ATF5), including conservative substitutions thereof. As usedherein, “conservative substitutions” are those amino acid substitutionswhich are functionally equivalent to a substituted amino acid residue,either because they have similar polarity or steric arrangement, orbecause they belong to the same class as the substituted residue (e.g.,hydrophobic, acidic, or basic). As described below, Westernimmunoblotting has permitted the identification of the major cellularform of ATF5 protein. The ATF5 cDNA sequence predicts two potentialin-frame methionine start sites that would lead to proteins ofapproximately 30 and 20 kDa. Observation that the major form of ATF5 incells has an apparent molecular mass of 20-22 kDa indicates favoredutilization of the second site. When a canonical Kozak initiationconsensus sequence was included upstream of the first methionine, thelarger protein was expressed, thereby indicating that the 22-kDa form isnot formed by cleavage of a 30-kDa precursor. Accordingly, the ATF5protein of the present invention further includes both the 22-kDa and30-kDa isomers thereof.

An “ATF5 analogue”, as used herein, is a functional variant of the ATF5protein, having ATF5 biological activity, that has 60% or greater (incertain embodiments, 70% or greater or 80% or greater or 90% or greateror 95% or greater) amino-acid-sequence homology with the ATF5 protein.As further used herein, the term “ATF5 biological activity” refers tothe activity of an ATF5 protein or ATF5 analogue to associate physicallywith, or bind with, CRE (i.e., binding of approximately two fold, or,more preferably, approximately five fold, above the background bindingof a negative control), under the conditions of the assays describedherein, although affinity may be different from that of native ATF5.

The skilled practitioner understands that the numbering of amino acidresidues in ATF5 may be different than that set forth herein, or maycontain certain conservative amino acid substitutions that produce thesame ATF5-CRE associating activity as that described herein.Corresponding amino acids and conservative substitutions in otherisoforms or analogues are easily identified by visually inspecting therelevant amino acid sequences, or by using commercially availablehomology software programs.

As outlined in the Examples section, interference with the functionand/or activity of ATF5 promote apoptosis of glioblastoma multiformetumors (GBM) in vitro and in vivo. Furthermore, selective interferencewith ATF5 function and/or activity in other carcinoma types is shown totriggers cell death. Culture and animal studies also show that thetranscription factor ATF5 is required for survival of GBM cells and thatlimited subcutaneous treatment with a CP-d/n-ATF5 causes apparent tumoreradication in a mouse model of endogenous gliomas without apparenttoxicity or side effects. As highlighted in the attached examples, theeffect of such ATF5 interference by administration of a CP-d/n-ATF5 isindeed specific, in that interfering with ATF5 function and/or activitytriggers increased cell death in neoplastic cells, but not normal cells.

As used herein, “dominant-negative ATF5” or “d/n-ATF5” is a peptidecomprising a portion of the human ATF5 amino acid sequence. In certainembodiments, the d/n-ATF5 peptide comprises the sequenceLEQENAELEGECQGLEARNRELKERAES (SEQ ID NO: 5), where the underlinedsequence is the dominant-negative sequence and the remainder of thesequence is the ATF5 leucine zipper. In certain embodiments the d/n-ATF5is encoded by a nucleic acid comprising the sequenceCTGGAACAGGAAAACGCGGAACTGGAAGGCGAATGCCAGGGCCTGGAAGCGCGCAACCGCGAACTGAAAGAACGCGCGGAAAGCTAA (SEQ ID NO: 20). In certainembodiments the d/n-ATF5 peptide comprises

(SEQ ID NO: 6) LEKEAEELEQENAELEGECQGLEARNRELKERAES.In certain embodiments the d/n-ATF5 is encoded by a nucleic acidcomprising the sequenceCTGGAAAAAGAAGCGGAAGAACTGGAACAGGAAAACGCGGAACTGGAAGGCGAATGCCAGGGCCTGGAAGCGCGCAACCGCGAACTGAAAGAACGCGCGGA AAGCTAA (SEQ ID NO:21). In certain embodiments, the d/n-ATF5 peptide comprises the sequence

(SEQ ID NO: 7) LARENEELLEKEA EELEQENAELEGECQGLEARNRELKERAES,where the underlined sequence is the dominant-negative sequence and theremainder of the sequence is the ATF5 leucine zipper. In certainembodiments the d/n-ATF5 is encoded by a nucleic acid comprising thesequence CTGGCGCGCGAAAACGAAGAACTGCTGGAAAAAGAAGCGGAAGAACTGGAACAGGAAAACGCGGAACTGGAAGGCGAATGCCAGGGCCTGGAAGCGCGCAACCGCGAACTGAAAGAACGCGCGGAAAGCTAA (SEQ ID NO: 22). In certain embodiments,the d/n-ATF5 peptide comprises the sequence

(SEQ ID NO: 23) LEQRAEELARENEELLEKEAEELEQENAELEGECQGLEARN RELKER AES,where the underlined sequence is the dominant-negative sequence and theremainder of the sequence is the ATF5 leucine zipper. In certainembodiments the d/n-ATF5 is encoded by a nucleic acid comprising thesequence CTGGAACAGCGCGCGGAAGAACTGGCGCGCGAAAACGAAGAACTGCTGGAAAAAGAAGCGGAAGAACTGGAACAGGAAAACGCGGAACTGGAAGGCGAATGCCAGGGCCTGGAAGCGCGCAACCGCGAACTGAAAGAACGCGCGGAAAGCTAA (SEQ ID NO: 24). In certainembodiments, the d/n-ATF5 peptide comprises the sequence

(SEQ ID NO: 9) LEQRAEELARENEELLEKEAEELEQENAELEGECQGLEARNREL KERA ESV,where the underlined sequence is the dominant-negative sequence and theremainder of the sequence is the ATF5 leucine zipper. In certainembodiments the d/n-ATF5 is encoded by a nucleic acid comprising thesequence CTGGAACAGCGCGCGGAAGAACTGGCGCGCGAAAACGAAGAACTGCTGGAAAAAGAAGCGGAAGAACTGGAACAGGAAAACGCGGAACTGGAAGGCGAATGCCAGGGCCTGGAAGCGCGCAACCGCGAACTGAAAGAACGCGCGGAAAGCGTGTAA (SEQ ID NO: 25). In certain embodiments, ad/n-ATF5 comprising the ATF5 leucin zipper sequenceLEGECQGLEARNRELKERAESV (SEQ ID NO: 26), will further comprise, 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, or 24 additional C-terminal ATF5 leucine zipper residues. In certainembodiments, the d/n-ATF5 peptide consists of one of the foregoingpeptide sequences.

As used herein, “dominant-negative ATF5” or “d/n-ATF5” is a peptidecomprising a portion of the rat or mouse ATF5 amino acid sequence. Incertain embodiments, the d/n-ATF5 peptide comprises the sequenceLEQENAELEGECQGLEARNRELRERAES (SEQ ID NO: 10), where the underlinedsequence is the dominant-negative sequence and the remainder of thesequence is the ATF5 leucine zipper. In certain embodiments the d/n-ATF5is encoded by a nucleic acid comprising the sequenceCTGGAACAGGAAAACGCGGAACTGGAAGGCGAATGCCAGGGCCTGGAAGCGCGCAACCGCGAACTGCGCGAACGCGCGGAAAGCTAA (SEQ ID NO: 27). Incertain embodiments, the d/n-ATF5 peptide comprises the sequence

(SEQ ID NO: 11) LEKEAEELEQENAELEGECQ GLEARNRELRERAES,where the underlined sequence is the dominant-negative sequence and theremainder of the sequence is the ATF5 leucine zipper. In certainembodiments the d/n-ATF5 is encoded by a nucleic acid comprising thesequence CTGGAAAAAGAAGCGGAAGAACTGGAACAGGAAAACGCGGAACTGGAAGGCGAATGCCAGGGCCTGGAAGCGCGCAACCGCGAACTGCGCGAACGCGCGGAAAGCTAA (SEQ ID NO: 28). In certainembodiments, the d/n-ATF5 peptide comprises the sequence

(SEQ ID NO: 12) LARENEELLEKEAEELEQENAELEGECQGL EARNRELRERAES,where the underlined sequence is the dominant-negative sequence and theremainder of the sequence is the ATF5 leucine zipper. In certainembodiments the d/n-ATF5 is encoded by a nucleic acid comprising thesequence CTGGCGCGCGAAAACGAAGAACTGCTGGAAAAAGAAGCGGAAGAACTGGAACAGGAAAACGCGGAACTGGAAGGCGAATGCCAGGGCCTGGAAGCGCGCAACCGCGAACTGCGCGAACGCGCGG AAAGCTAA (SEQ ID NO:29). In certain embodiments, the d/n-ATF5 peptide comprises the sequence

(SEQ ID NO: 13) LEQRAEELARENEELLEKEAEELEQENAELEGECQGLEARNRELRER AES,where the underlined sequence is the dominant-negative sequence and theremainder of the sequence is the ATF5 leucine zipper. In certainembodiments the d/n-ATF5 is encoded by a nucleic acid comprising thesequence CTGGAACAGCGCGCGGAAGAACTGGCGCGCGAAAACGAAGAACTGCTGGAAAAAGAAGCGGAAGAACTGGAACAGGAAAACGCGGAACTGGAAGGCGAATGCCAGGGCCTGGAAGCGCGCAACCGCGAACTGCGCGAACGC GCGGAAAGCTAA (SEQ IDNO: 30). In certain embodiments, the d/n-ATF5 peptide comprises thesequence

(SEQ ID NO: 14) LEQRAEELARENEELLEKEAEELEQENAELEGECQGLEARNRELRER AESV,where the underlined sequence is the dominant-negative sequence and theremainder of the sequence is the ATF5 leucine zipper. In certainembodiments the d/n-ATF5 is encoded by a nucleic acid comprising thesequence CTGGAACAGCGCGCGGAAGAACTGGCGCGCGAAAACGAAGAACTGCTGGAAAAAGAAGCGGAAGAACTGGAACAGGAAAACGCGGAACTGGAAGGCGAATGCCAGGGCCTGGAAGCGCGCAACCGCGAACTGCGCGAACGCGC GGAAAGCGTGTAA (SEQID NO: 31). In certain embodiments, a d/n-ATF5 comprising the ATF5leucin zipper sequence LEGECQGLEARNRELRERAESV (SEQ ID NO: 32), willfurther comprise, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, or 24 additional C-terminal ATF5 leucinezipper residues of the sequence EREIQYVKDLLIEVYKARSQRTRS (SEQ ID NO:33). In certain embodiments, the d/n-ATF5 peptide consists of one of theforegoing peptide sequences.

As used herein, a “cell-penetrating peptide” or “CP” is a peptide thatcomprises a short amino acid sequence (e.g., in certain embodiments,about 12-30 residues) or functional motif that confers theenergy-independent (i.e., non-endocytotic) translocation propertiesassociated with transport of the membrane-permeable complex across theplasma and/or nuclear membranes of a cell. Representative amino acidmotifs conferring such properties are listed in U.S. Pat. No. 6,348,185,the contents of which are expressly incorporated herein by reference.The cell-penetrating peptides of the present invention preferablyinclude, but are not limited to, penetratin1, transportan, pIsl,TAT(48-60), pVEC, MTS, and MAP.

The cell-penetrating peptides of the present invention include thosesequences that retain certain structural and functional features of theidentified cell-penetrating peptides, yet differ from the identifiedpeptides' amino acid sequences at one or more positions. Suchpolypeptide variants can be prepared by substituting, deleting, oradding amino acid residues from the original sequences via methods knownin the art.

In certain embodiments, such substantially similar sequences includesequences that incorporate conservative amino acid substitutions, asdescribed above in connection with polypeptide apoptotic targetinhibitors. In certain embodiments, a cell-penetrating peptide of thepresent invention is at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%,77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homologous to the aminoacid sequence of the identified peptide and is capable of mediating cellpenetration. The effect of the amino acid substitutions on the abilityof the synthesized peptide to mediate cell penetration can be testedusing the methods disclosed in Examples section, below.

In certain embodiments of the present invention, the cell-penetratingpeptide is penetratin1, comprising the peptide sequence RQIKIWFQNRRMKWKK(SEQ ID NO: 34), or a conservative variant thereof. As used herein, a“conservative variant” is a peptide having one or more amino acidsubstitutions, wherein the substitutions do not adversely affect theshape—or, therefore, the biological activity (i.e., transport activity)or membrane toxicity—of the cell-penetrating peptide.

Penetratin1 is a 16-amino-acid polypeptide derived from the thirdalpha-helix of the homeodomain of Drosophila antennapedia. Its structureand function have been well studied and characterized: Derossi et al.,Trends Cell Biol., 8(2):84-87, 1998; Dunican et al., Biopolymers,60(1):45-60, 2001; Hallbrink et al., Biochim. Biophys. Acta,1515(2):101-09, 2001; Bolton et al., Eur. J. Neurosci., 12(8):2847-55,2000; Kilk et al., Bioconjug. Chem., 12(6):911-16, 2001; Bellet-Amalricet al., Biochim. Biophys. Acta, 1467(1):131-43, 2000; Fischer et al., J.Pept. Res., 55(2): 163-72, 2000; Thoren et al., FEBS Lett.,482(3):265-68, 2000.

It has been shown that penetratin1 efficiently carries avidin, a 63-kDaprotein, into human Bowes melanoma cells (Kilk et al., Bioconjug. Chem.,12(6):911-16, 2001). Additionally, it has been shown that thetransportation of penetratin1 and its cargo is non-endocytotic andenergy-independent, and does not depend upon receptor molecules ortransporter molecules. Furthermore, it is known that penetratin1 is ableto cross a pure lipid bilayer (Thoren et al., FEBS Lett., 482(3):265-68,2000). This feature enables penetratin1 to transport its cargo, freefrom the limitation of cell-surface-receptor/-transporter availability.The delivery vector previously has been shown to enter all cell types(Derossi et al., Trends Cell Biol., 8(2):84-87, 1998), and effectivelyto deliver peptides (Troy et al., Proc. Natl. Acad. Sci. USA,93:5635-40, 1996) or antisense oligonucleotides (Troy et al., J.Neurosci., 16:253-61, 1996; Troy et al., J. Neurosci., 17:1911-18,1997).

In certain embodiments, the CP-d/n-ATF5 is a peptide comprising aPenetratin sequence operably linked to a rat d/n-ATF5 sequence. Incertain embodiments the CP-d/n-ATF5 peptide sequence is

(SEQ ID NO: 15) MGSSHHHHHHSSGLVPRGSHM RQIKIWFQNRRMKWKK DYKDDDDKMASMTGGQQMGRDPD

LEGECQG LEARNRELRERAESV ,where the underlined residues are a 6XHis-tag leader sequence (“6XHis”disclosed as SEQ ID NO: 16), the bold residues are a Penetratinsequence, the italicized residues are a Flag tag, the residues with nofont modification are spacer amino acids, the bold and italicizedresidues are a d/n sequence, and the bold and underlined residues are anATF5 leucine zipper truncated after its first Valine. In certainembodiments the CP-d/n-ATF5 is encoded by a nucleic acid comprising thesequence ATGGGCAGCAGCCATCATCATCATCATCACAGCAGCGGCCTGGTGCCGCGCGGCAGCCATATGCGTCAAATTAAAATTTGGTTTCAAAATCGTCGTATGAAATGGAAAAAAGACTACAAGGACGATGATGACAAAATGGCATCTATGACTGGAGGACAACAAATGGGAAGAGACCCAGACCTCGAACAAAGAGCAGAAGAACTAGCAAGAGAAAACGAAGAACTACTAGAAAAAGAAGCAGAAGAACTAGAACAAGAAAATGCAGAGCTAGAGGGCGAGTGCCAAGGGCTAGAGGCGCGGAATCGGGAGCTGAGGGAGAGGGCAGAGTCAGTGTAG (SEQ ID NO: 35).

Other non-limiting embodiments of the present invention involve the useof the following exemplary cell permeant molecules: RL16(H-RRLRRLLRRLLRRLRR-OH (SEQ ID NO: 36)), a sequence derived fromPenetratin1 with slightly different physical properties (Biochim BiophysActa. 2008 July-August; 1780(7-8):948-59); and RVGRRRRRRRRR (SEQ ID NO:37), a rabies virus sequence which targets neurons see P. Kumar, H. Wu,J. L. McBride, K. E. Jung, M. H. Kim, B. L. Davidson, S. K. Lee, P.Shankar and N. Manjunath, Transvascular delivery of small interferingRNA to the central nervous system, Nature 448 (2007), pp. 39-43.

In certain alternative non-limiting embodiments of the presentinvention, the cell-penetrating peptide is a cell-penetrating peptidesselected from the group consisting of: transportan, pISl, Tat(48-60),pVEC, MAP, and MTS. Transportan is a 27-amino-acid long peptidecontaining 12 functional amino acids from the amino terminus of theneuropeptide galanin, and the 14-residue sequence of mastoparan in thecarboxyl terminus, connected by a lysine (Pooga et al., FASEB J.,12(1):67-77, 1998). It comprises the amino acid sequenceGWTLNSAGYLLGKINLKALAALAKKIL (SEQ ID NO: 38), or a conservative variantthereof.

pIsl is derived from the third helix of the homeodomain of the ratinsulin 1 gene enhancer protein (Magzoub et al., Biochim. Biophys. Acta,1512(1):77-89, 2001; Kilk et al., Bioconjug. Chem., 12(6):911-16, 2001).pIsl comprises the amino acid sequence PVIRVW FQNKRCKDKK (SEQ ID NO:39), or a conservative variant thereof.

Tat is a transcription activating factor, of 86-102 amino acids, thatallows translocation across the plasma membrane of an HIV-infected cell,to transactivate the viral genome (Hallbrink et al., Biochem. Biophys.Acta., 1515(2):101-09, 2001; Suzuki et al., J. Biol. Chem.,277(4):2437-43, 2002; Futaki et al., J. Biol. Chem., 276(8):5836-40,2001). A small Tat fragment, extending from residues 48-60, has beendetermined to be responsible for nuclear import (Vives et al., J. Biol.Chem., 272(25):16010-017, 1997); it comprises the amino acid sequence:YGRKKRRQRRR (SEQ ID NO: 40); GRKKRRQRRRPPQ (SEQ ID NO: 41); or aconservative variant thereof.

In certain embodiments, the CP-d/n-ATF5 is a peptide comprising a TATsequence operably linked to a rat d/n-ATF5 sequence. In certainembodiments the CP-d/n-ATF5 peptide sequence is

(SEQ ID NO: 17) MGSSHHHHHHSSGLVPRGSHMLE YGRKKRRQRRR YPYDVPDYAMASMTGGQQMGRDPD

LEGECQGLE ARNRELRERAESV ,where the underlined residues are a 6XHis-tag leader sequence (“6XHis”disclosed as SEQ ID NO: 16), the bold residues are a TAT sequence, theitalicized residues are an HA tag, the residues with no fontmodification are spacer amino acids, the bold and italicized residuesare a d/n sequence, and the bold and underlined residues are an ATF5leucine zipper truncated after its first Valine. In certain embodimentsthe CP-d/n-ATF5 is encoded by a nucleic acid comprising the sequenceATGGGCAGCAGCCATCATCATCATCATCACAGCAGCGGCCTGGTGCCGCGCGGCAGCCATATGCTCGAGTACGGCCGCAAGAAACGCCGCCAGCGCCGCCGCTATCCATATGACGTCCCAGACTATGCTATGGCATCTATGACTGGAGGACAACAAATGGGAAGAGACCCAGACCTCGAACAAAGAGCAGAAGAACTAGCAAGAGAAAACGAAGAACTACTAGAAAAAGAAGCAGAAGAACTAGAACAAGAAAATGCAGAGCTAGAGGGCGAGTGCCAAGGGCTAGAGGCGCGGAATCGGGAGCTGAGGGAGAGGGCAGAGTCAGTGTAG (SEQ ID NO: 42).

pVEC is an 18-amino-acid-long peptide derived from the murine sequenceof the cell-adhesion molecule, vascular endothelial cadherin, extendingfrom amino acid 615-632 (Elmquist et al., Exp. Cell Res., 269(2):237-44,2001). pVEC comprises the amino acid sequence LLIILRRRIRKQAHAH (SEQ IDNO: 43), or a conservative variant thereof.

MTSs, or membrane translocating sequences, are those portions of certainpeptides which are recognized by the acceptor proteins that areresponsible for directing nascent translation products into theappropriate cellular organelles for further processing (Lindgren et al.,Trends in Pharmacological Sciences, 21(3):99-103, 2000; Brodsky, J. L.,Int. Rev. Cyt., 178:277-328, 1998; Zhao et al., J. Immunol. Methods,254(1-2):137-45, 2001). An MTS of particular relevance is MPS peptide, achimera of the hydrophobic terminal domain of the viral gp41 protein andthe nuclear localization signal from simian virus 40 large antigen; itrepresents one combination of a nuclear localization signal and amembrane translocation sequence that is internalized independent oftemperature, and functions as a carrier for oligonucleotides (Lindgrenet al., Trends in Pharmacological Sciences, 21(3):99-103, 2000; Morriset al., Nucleic Acids Res., 25:2730-36, 1997). MPS comprises the aminoacid sequence GALFLGWLGAAGSTMGAWSQPKKKRKV (SEQ ID NO: 44), or aconservative variant thereof.

Model amphipathic peptides, or MAPs, form a group of peptides that have,as their essential features, helical amphipathicity and a length of atleast four complete helical turns (Scheller et al., J. Peptide Science,5(4):185-94, 1999; Hallbrink et al., Biochim. Biophys. Acta.,1515(2):101-09, 2001). An exemplary MAP comprises the amino acidsequence KLALKLALKALKAALKLA-amide (SEQ ID NO: 45), or a conservativevariant thereof.

In certain embodiments, the cell-penetrating peptides described aboveare covalently bound to the d/n-ATF5, e.g., via a peptide bond. Incertain embodiments the cell-penetrating peptide is operably linked to ad/n-ATF5 via recombinant DNA technology. For example, the d/n-ATF5 canbe introduced either upstream (for linkage to the amino terminus of thecell-penetrating peptide) or downstream (for linkage to the carboxyterminus of the cell-penetrating peptide), or both, of a nucleic acidsequence encoding the cell-penetrating peptide of interest. Such fusionsequences comprising both the d/n-ATF5 encoding nucleic acid sequenceand the cell-penetrating peptide encoding nucleic acid sequence can beexpressed using techniques well known in the art.

In certain embodiments the d/n-ATF5 can be operably linked to thecell-penetrating peptide via a non-covalent linkage. In certainembodiments such non-covalent linkage is mediated by ionic interactions,hydrophobic interactions, hydrogen bonds, or van der Waals forces.

In certain embodiments the d/n-ATF5 is operably linked to the cellpenetrating peptide via a chemical linker. Examples of such linkagestypically incorporate 1-30 nonhydrogen atoms selected from the groupconsisting of C, N, O, S and P. Exemplary linkers include, but are notlimited to, a substituted alkyl or a substituted cycloalkyl.Alternately, the heterologous moiety may be directly attached (where thelinker is a single bond) to the amino or carboxy terminus of thecell-penetrating peptide. When the linker is not a single covalent bond,the linker may be any combination of stable chemical bonds, optionallyincluding, single, double, triple or aromatic carbon-carbon bonds, aswell as carbon-nitrogen bonds, nitrogen-nitrogen bonds, carbon-oxygenbonds, sulfur-sulfur bonds, carbon-sulfur bonds, phosphorus-oxygenbonds, phosphorus-nitrogen bonds, and nitrogen-platinum bonds. Incertain embodiments, the linker incorporates less than 20 nonhydrogenatoms and are composed of any combination of ether, thioether, urea,thiourea, amine, ester, carboxamide, sulfonamide, hydrazide bonds andaromatic or heteroaromatic bonds. In certain embodiments, the linker isa combination of single carbon-carbon bonds and carboxamide, sulfonamideor thioether bonds.

A general strategy for conjugation involves preparing thecell-penetrating peptide and the d/n-ATF5 components separately, whereineach is modified or derivatized with appropriate reactive groups toallow for linkage between the two. The modified d/n-ATF5 is thenincubated together with a cell-penetrating peptide that is prepared forlinkage, for a sufficient time (and under such appropriate conditions oftemperature, pH, molar ratio, etc.) as to generate a covalent bondbetween the cell-penetrating peptide and the d/n-ATF5.

The present invention contemplates the use of proteins and proteinanalogues generated by synthesis of polypeptides in vitro, e.g., bychemical means or in vitro translation of mRNA. For example, ATF5 andinhibitors thereof may be synthesized by methods commonly known to oneskilled in the art (Modern Techniques of Peptide and Amino Acid Analysis(New York: John Wiley & Sons, 1981); Bodansky, M., Principles of PeptideSynthesis (New York: Springer-Verlag New York, Inc., 1984). Examples ofmethods that may be employed in the synthesis of the amino acidsequences, and analogues of these sequences, include, but are notlimited to, solid-phase peptide synthesis, solution-method peptidesynthesis, and synthesis using any of the commercially-available peptidesynthesizers. The amino acid sequences of the present invention maycontain coupling agents and protecting groups, which are used in thesynthesis of protein sequences, and which are well known to one of skillin the art.

As used herein, “amino acid residue,” “amino acid,” or “residue,”includes genetically encoded amino acid residues and non-geneticallyencoded amino acid residues, e.g., non-genetically encoded amino acidresidues or non-natural amino acids include, but are not limited toD-enantiomers of naturally occurring chiral amino acids, β-alanine(β-Ala); 2,3-diaminopropionic acid (Dpr); nipecotic acid (Nip);pipecolic acid (Pip); ornithine (Orn); citrulline (Cit); t-butylalanine(t-BuA); 2-t-butylglycine (t-BuG); N-methylisoleucine (MeIle);phenylglycine (PhG); cyclohexylalanine (ChA); norleucine (Nle);naphthylalanine (Nal); 4-chlorophenylalanine (Phe(4-Cl));2-fluorophenylalanine (Phe(2-F)); 3-fluorophenylalanine (Phe(3-F));4-fluorophenylalanine (Phe(4-F)); penicillamine (Pen);1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic);β-2-thienylalanine (Thi); methionine sulfoxide (MSO); homoarginine(hArg); N-acetyl lysine (AcLys); 2,4-diaminobutyric acid (Dbu);2,3-diaminobutyric acid (Dab); p-aminophenylalanine (Phe (pNH2));N-methyl valine (MeVal); homocysteine (hCys), homophenylalanine (hPhe);homoserine (hSer); hydroxyproline (Hyp); homoproline (hPro); and thecorresponding D-enantiomer of each of the foregoing, e.g., D-β-Ala,D-Dpr, D-Nip, D-Orn, D-Cit, D-t-BuA, D-t-BuG, D-MeIle, D-PhG, D-ChA,D-Nle, D-NaI, D-Phe(4-Cl), D-Phe(2-F), D-Phe(3-F), D-Phe(4-F), D-Pen,D-Tic, D-Thi, D-MSO, D-hArg, D-AcLys, D-Dbu, D-Dab, D-Phe(pNH2),D-MeVal, D-hCys, D-hPhe, D-hSer, D-Hyp, and D-hPro. Additionalnon-genetically encoded amino acid residues include 3-aminopropionicacid; 4-aminobutyric acid; isonipecotic acid (Inp); aza-pipecolic acid(azPip); aza-proline (azPro); α-aminoisobutyric acid (Aib);ε-aminohexanoic acid (Aha); δ-aminovaleric acid (Ava); N-methylglycine(MeGly).

In certain embodiments, the cell-penetrating dominant-negative ATF5comprises a sequence selected from the group consisting of: (1)

(SEQ ID NO: 15) MGSSHHHHHHSSGLVPRGSHM RQIKIWFQNRRMKWKK DYKDDDDKMASMTGGQQMGRDPD

LEGECQG LEARNRELRERAES V ,where the underlined residues (MG-HM) are a 6XHis-tag leader sequence(“6XHis” disclosed as SEQ ID NO: 16), the bold residues

are a Penetratin sequence, the italicized residues (DY-DK) are a Flagtag, the residues with no font modification (MA-PD) are spacer aminoacids, the bold and italicized residues

are a d/n sequence, and the bold and underlined residues

are an ATF5 leucine zipper truncated after its first Valine; (2)

(SEQ ID NO: 17) MGSSHHHHHHSSGLVPRGSHMLE YGRKKRRQRRR YPYDVPDYAMASMTGGQQMGRDPD

LEGECQGLE ARNRELRERAESV ,where the underlined residues (MG-LE) are a 6XHis-tag leader sequence(“6XHis” disclosed as SEQ ID NO: 16), the bold residues

are a TAT sequence, the italicized residues (YP-YA) are an HA tag, theresidues with no font modification (MA-PD) are spacer amino acids, thebold and italicized residues

are a d/n sequence, and the bold and underlined residues

are an ATF5 leucine zipper truncated after its first Valine; (3)

(SEQ ID NO: 18) MGSSHHHHHHSSGLVPRGSHM RQIKIWFQNRRMKWKK LEQRAEELARENEELLEKEAEELEQENAE LEGECQGLEARNRELKER AESVwhere the where the underlined residues (MG-HM) are a 6XHis-tag leadersequence (“6XHis” disclosed as SEQ ID NO: 16), the bold residues

are a Penetratin sequence, the italicized residues (LE-AE) are a d/nsequence, and the bold and underlined residues

are an ATF5 leucine zipper truncated after its first Valine; and (4)

(SEQ ID NO: 19) RQIKIWRQNRRMKWKK LEQRAEELARENEELLEKEAEELEQENAE LEGECQGLEARNRELKERAESVwhere the bold residues

are a Penetratin sequence, the italicized residues (LE-AE) are a d/nsequence, and the bold and underlined residues

are an ATF5 leucine zipper truncated after its first Valine. In certainembodiments, the cell-penetrating dominant-negative ATF5 is chemicallysynthesized.

Use of D/N-ATF5 Compositions

In accordance with methods described herein, ATF5 can be inhibited in acell by disabling, disrupting, or inactivating the function or activityof ATF5 in the cell. For example, the function or activity of ATF5 in acell may be inhibited by providing a dominant negative-ATF5 moleculecapable of inhibiting the function or activity of native ATF5 in thecell. In certain embodiments, the d/n-ATF5 is a CP-d/n-ATF5.

In certain embodiments, function or activity of the ATF5 in the cell isinhibited by at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, or greater(inclusive of intermediate ranges between those explicitly recited,e.g., 5-10%, 10-20%, 20-30%, 40-50%, or greater than 50% including50%-100%). In certain embodiments, function or activity of the ATF5 isdecreased by inhibiting expression of ATF5. Such expression can beinhibited by at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, or greater(inclusive of intermediate ranges between those explicitly recited,e.g., 5-10%, 10-20%, 20-30%, 40-50%, or greater than 50% including50%-100%). In certain embodiments, expression is decreased by 60%, 80%,or 90%, as outlined in FIG. 11A-11E.

In certain embodiments, the present invention provides methods fortreating or preventing a tumor in a subject in need of treatment,comprising administering to the subject a pharmaceutical compositioncomprising a CP-d/n-ATF5 and, optionally, a pharmaceutically-acceptablecarrier. The CP-d/n-ATF5 is provided in an amount that is effective totreat the tumor in a subject to whom the composition is administered. Asused herein, the phrase “effective” means effective to ameliorate orminimize the clinical impairment or symptoms of the tumor. For example,the clinical impairment or symptoms of the tumor may be ameliorated orminimized by diminishing any pain or discomfort suffered by the subject;by extending the survival of the subject beyond that which wouldotherwise be expected in the absence of such treatment; by inhibiting orpreventing the development or spread of the tumor; or by limiting,suspending, terminating, or otherwise controlling the maturation andproliferation of cells in the tumor. The amount of CP-d/n-ATF5 effectiveto treat a tumor in a subject in need of treatment will vary dependingupon the particular factors of each case, including the type of tumor,the stage of the tumor, the subject's weight, the severity of thesubject's condition, and the method of administration. This amount canbe readily determined by the skilled artisan.

As used herein, the term “tumor” refers to a pathologic proliferation ofcells, and includes a neoplasia. The term “neoplasia”, and related termsas further used herein, refers to the uncontrolled and progressivemultiplication of tumor cells under conditions that would not elicit, orwould cause cessation of, multiplication of normal cells. Neoplasiaresults in the formation of a “neoplasm”, which is defined herein tomean any new and abnormal growth, particularly a new growth of tissue,in which the growth of cells is uncontrolled and progressive. As usedherein, neoplasms include, without limitation, morphologicalirregularities in cells in tissue of a subject, as well as pathologicproliferation of cells in tissue of a subject, as compared with normalproliferation in the same type of tissue. Additionally, neoplasmsinclude benign tumors and malignant tumors. Malignant neoplasms aredistinguished from benign in that the former show a greater degree ofanaplasia, or loss of differentiation and orientation of cells, and havethe properties of invasion and metastasis. Thus, neoplasia includes“cancer” including hematological cancers, which herein refers to aproliferation of tumor cells having the unique trait of loss of normalcontrols, resulting in unregulated growth, lack of differentiation,local tissue invasion, and metastasis.

Additionally, as used herein, the term “neural tumor” refers to atumorigenic form of neural cells (i.e., transformed neural cells), andincludes astrocytoma cells (i.e., cells of all astrocytomas, including,without limitation, Grades I-IV astrocytomas, anaplastic astrocytoma,astroblastoma, astrocytoma fibrillare, astrocytoma protoplasmaticum,gemistocytic astrocytoma, and glioblastoma multiforme), gliomas,medulloblastomas, neuroblastomas, and other brain tumors. Brain tumorsinvade and destroy normal tissue, producing such effects as impairedsensorimotor and cognitive function, increased intracranial pressure,cerebral edema, and compression of brain tissue, cranial nerves, andcerebral vessels. Metastases may involve the skull or any intracranialstructure. The size, location, rate of growth, and histologic grade ofmalignancy determine the seriousness of brain tumors. Nonmalignanttumors grow slowly, with few mitoses, no necrosis, and no vascularproliferation. Malignant tumors grow more rapidly, and invade othertissues. However, they rarely spread beyond the CNS, because they causedeath by local growth.

Brain tumors may be classified by site (e.g., brain stem, cerebellum,cerebrum, cranial nerves, ependyma, meninges, neuroglia, pineal region,pituitary gland, and skull) or by histologic type (e.g., meningioma,primary CNS lymphoma, or astrocytoma). Common primary childhood tumorsare cerebellar astrocytomas and medulloblastomas, ependymomas, gliomasof the brain stem, neuroblastomas, and congenital tumors. In adults,primary tumors include meningiomas, schwannomas, and gliomas of thecerebral hemispheres (particularly the malignant glioblastoma multiformeand anaplastic astrocytoma, and the more benign astrocytoma andoligodendroglioma). Overall incidence of intracranial neoplasms isessentially equal in males and females, but cerebellar medulloblastomaand glioblastoma multiforme are more common in males.

Gliomas are tumors composed of tissue representing neuroglia in any oneof its stages of development. They account for 45% of intracranialtumors. Gliomas can encompass all of the primary intrinsic neoplasms ofthe brain and spinal cord, including astrocytomas, ependymomas, andneurocytomas. Astrocytomas are tumors composed of transformedastrocytes, or astrocytic tumor cells. Such tumors have been classifiedin order of increasing malignancy: Grade I consists of fibrillary orprotoplasmic astrocytes; Grade II is an astroblastoma, consisting ofcells with abundant cytoplasm and two or three nuclei; and Grades IIIand IV are forms of glioblastoma multiforme, a rapidly growing tumorthat is usually confined to the cerebral hemispheres and composed of amixture of astrocytes, spongioblasts, astroblasts, and other astrocytictumor cells. Astrocytoma, a primary CNS tumor, is frequently found inthe brain stem, cerebellum, and cerebrum. Anaplastic astrocytoma andglioblastoma multiforme are commonly located in the cerebrum. Thepresent invention additionally provides methods for promoting apoptosisin a neoplastic cell comprising contacting the neoplastic cell with anATF5 inhibitor. The neoplastic cell can be selected from the groupconsisting of: breast, ovary, endometrium, gastric, colon, liver,pancreas, kidney, bladder, prostate, testis, skin, esophagus, tongue,mouth, parotid, larynx, pharynx, lymph node, lung, and brain. In oneembodiment, the neoplastic cell is selected from the group consisting ofglioblastoma, astrocytoma, glioma, medulloblastoma and neuroblastoma.

For example, but not by way of limitation, cell lines shown throughtesting to be susceptible to TAT-d/n-ATF5 (1-3 μM range) include: U87(human glioblastoma); U373 (human glioblastoma); LN229 (humanglioblastoma); C6 (rat glioblastoma); Mel501 (human melanoma); H2452(human mesothelioma); MDA-MB-468 (human breast cancer). In addition, anon-limiting list of cell lines shown through testing to be susceptibleto PEN-d/n-ATF5 (3 μM) include: Panc-1 (human pancreatic cancer);SH-SY5Y (human neuroblastoma cells); and HCT-116 (colon-carcinomacancer). The method of the present invention can be performed in vitroas well as in vivo in a subject. As used herein, “apoptosis” refers tocell death which is wholly or partially genetically controlled.

As outlined in the examples below, certain CP-d/n-ATF5 compositions areeffective anti-neoplastic agents across species, e.g., rat/mouseCP-d/n-ATF5 is effective against human cancers. Thus, in certainembodiments the CP-d/n-ATF5 can comprise a rat or mouse d/n-ATF5 peptidesequence and the subject may be any animal, including, but not limitedto a mammal (e.g., a human, domestic animal, or commercial animal). Incertain embodiments, the CP-d/n-ATF5 can comprise a rat or moused/n-ATF5 peptide sequence and the subject is a human.

In accordance with the method of the present invention, CP-d/n-ATF5 canbe administered to a human or animal subject by known procedures,including, without limitation, oral administration, parenteraladministration, intranasal administration and transdermaladministration. Preferably, the inhibitors or factors are administeredparenterally, by intracranial, intraspinal, intrathecal, or subcutaneousinjection.

D/N-ATF5 Pharmaceutical Compositions

For oral administration, CP-d/n-ATF5 can be formulated as capsules,tablets, powders, granules, or as a suspension. The CP-d/n-ATF5formulation may have conventional additives, such as lactose, mannitol,corn starch, or potato starch. The CP-d/n-ATF5 formulation also may bepresented with binders, such as crystalline cellulose, cellulosederivatives, acacia, corn starch, or gelatins. Additionally, theCP-d/n-ATF5 formulation may be presented with disintegrators, such ascorn starch, potato starch, or sodium carboxymethylcellulose. TheCP-d/n-ATF5 formulation also may be presented with dibasic calciumphosphate anhydrous or sodium starch glycolate. Finally, the CP-d/n-ATF5formulation may be presented with lubricants, such as talc or magnesiumstearate.

For parenteral administration (i.e., administration by injection througha route other than the alimentary canal), CP-d/n-ATF5 can be combinedwith a sterile aqueous solution that is preferably isotonic with theblood of the subject. Such a CP-d/n-ATF5 formulation can be prepared bydissolving a solid active ingredient in water containingphysiologically-compatible substances, such as sodium chloride, glycine,and the like, and having a buffered pH compatible with physiologicalconditions, so as to produce an aqueous solution, then rendering saidsolution sterile. The CP-d/n-ATF5 formulation can be presented in unitor multi-dose containers, such as sealed ampoules or vials. TheCP-d/n-ATF5 formulation can be delivered by any mode of injection,including, without limitation, epifascial, intracapsular, intracranial,intracutaneous, intrathecal, intramuscular, intraorbital,intraperitoneal, intraspinal, intrasternal, intravascular, intravenous,parenchymatous, subcutaneous, or sublingual.

In certain embodiments, the CP-d/n-ATF5 formulation is prepared forintranasal delivery. For nasal administration, solutions or suspensionscomprising the CP-d/n-ATF5 formulation can be prepared for directapplication to the nasal cavity by conventional means, for example witha dropper, pipette or spray. Other means for delivering the nasal spraycomposition, such as inhalation via a metered dose inhaler (MDI), mayalso be used according to the present invention. Several types of MDIsare regularly used for administration by inhalation. These types ofdevices can include breath-actuated MDI, dry powder inhaler (DPI),spacer/holding chambers in combination with MDI, and nebulizers. Theterm “MDI” as used herein refers to an inhalation delivery systemcomprising, for example, a canister containing an active agent dissolvedor suspended in a propellant optionally with one or more excipients, ametered dose valve, an actuator, and a mouthpiece. The canister isusually filled with a solution or suspension of an active agent, such asthe nasal spray composition, and a propellant, such as one or morehydrofluoroalkanes. When the actuator is depressed a metered dose of thesolution is aerosolized for inhalation. Particles comprising the activeagent are propelled toward the mouthpiece where they may then be inhaledby a subject. The formulations may be provided in single or multidoseform. For example, in the case of a dropper or pipette, this may beachieved by the patient administering an appropriate, predeterminedvolume of the solution or suspension. In the case of a spray, this maybe achieved for example by means of a metering atomising spray pump. Toimprove nasal delivery and retention the components according to theinvention may be encapsulated with cyclodextrins, or formulated withagents expected to enhance delivery and retention in the nasal mucosa.

Commercially available administration devices that are used or can beadapted for nasal administration of a composition of the inventioninclude the AERONEB™ (Aerogen, San Francisco, Calif.), AERONEB GO™(Aerogen); PARI LC PLUS™, PARI BOY™ N, PARI™ eflow (a nebulizerdisclosed in U.S. Pat. No. 6,962,151), PARI LC SINUS™, PARI SINUSTAR™,PARI SINUNEB™, VibrENT™ and PARI DURANEB™ (PART Respiratory Equipment,Inc., Monterey, Calif. or Munich, Germany); MICROAIR™ (Omron Healthcare,Inc, Vernon Hills, Ill.), HALOLITE™ (Profile Therapeutics Inc, Boston,Mass.), RESPIMAT™ (Boehringer Ingelheim, Germany), AERODOSE™ (Aerogen,Inc, Mountain View, Calif.), OMRON ELITE™ (Omron Healthcare, Inc, VernonHills, Ill.), OMRON MICROAIR™ (Omron Healthcare, Inc, Vernon Hills,Ill.), MABISMIST™ II (Mabis Healthcare, Inc, Lake Forest, Ill.),LUMISCOPE™ 6610, (The Lumiscope Company, Inc, East Brunswick, N.J.),AIRSEP MYSTIQUE™, (AirSep Corporation, Buffalo, N.Y.), ACORN-1™ andACORN-II™ (Vital Signs, Inc, Totowa, N.J.), AQUATOWER™ (MedicalIndustries America, Adel, Iowa), AVA-NEB™ (Hudson Respiratory CareIncorporated, Temecula, Calif.), AEROCURRENT™ utilizing the AEROCELL™disposable cartridge (AerovectRx Corporation, Atlanta, Ga.), CIRRUS™(Intersurgical Incorporated, Liverpool, N.Y.), DART™ (ProfessionalMedical Products, Greenwood, S.C.), DEVILBISS™ PULMO AIDE (DeVilbissCorp; Somerset, Pa.), DOWNDRAFT™ (Marquest, Englewood, Colo.), FAN JET™(Marquest, Englewood, Colo.), MB-5™ (Mefar, Bovezzo, Italy), MISTY NEB™(Baxter, Valencia, Calif.), SALTER 8900™ (Salter Labs, Arvin, Calif.),SIDESTREAM™ (Medic-Aid, Sussex, UK), UPDRAFT-II™ (Hudson RespiratoryCare; Temecula, Calif.), WHISPER JET™ (Marquest Medical Products,Englewood, Colo.), AIOLOS™ (Aiolos Medicinsk Teknik, Karlstad, Sweden),INSPIRON™ (Intertech Resources, Inc., Bannockburn, Ill.), OPTIMIST™(Unomedical Inc., McAllen, Tex.), PRODOMO™, SPIRA™ (Respiratory CareCenter, Hameenlinna, Finland), AERx™ Essence™ and Ultra™, (AradigmCorporation, Hayward, Calif.), SONIK™ LDI Nebulizer (Evit Labs,Sacramento, Calif.), ACCUSPRAY™ (BD Medical, Franklin Lake, N.J.),ViaNase ID™ (electronic atomizer; Kurve, Bothell, Wash.), OptiMist™device or OPTINOSE™ (Oslo, Norway), MAD Nasal™ (Wolfe Tory Medical,Inc., Salt Lake City, Utah), Freepod™ (Valois, Marly le Roi, France),Dolphin™ (Valois), Monopowder™ (Valois), Equadel™ (Valois), VP3™ andVP7™ (Valois), VP6 Pump™ (Valois), Standard Systems Pumps™ (Ing. ErichPfeiffer, Radolfzell, Germany), AmPump™ (Ing. Erich Pfeiffer), CountingPump™ (Ing. Erich Pfeiffer), Advanced Preservative Free System™ (Ing.Erich Pfeiffer), Unit Dose System™ (Ing. Erich Pfeiffer), Bidose System™(Ing. Erich Pfeiffer), Bidose Powder System™ (Ing. Erich Pfeiffer),Sinus Science™ (Aerosol Science Laboratories, Inc., Camarillo, Calif.),ChiSys™ (Archimedes, Reading, UK), Fit-Lizer™ (Bioactis, Ltd, a SNBLsubsidiary (Tokyo, J P), Swordfish V™ (Mystic Pharmaceuticals, Austin,Tex.), DirectHaler™ Nasal (DirectHaler, Copenhagen, Denmark) andSWIRLER™ Radioaerosol System (AMICI, Inc., Spring City, Pa.).

For transdermal administration, CP-d/n-ATF5 can be combined with skinpenetration enhancers, such as propylene glycol, polyethylene glycol,isopropanol, ethanol, oleic acid, N-methylpyrrolidone, and the like,which increase the permeability of the skin to the CP-d/n-ATF5, andpermit the CP-d/n-ATF5 to penetrate through the skin and into thebloodstream. The CP-d/n-ATF5 compositions also may be further combinedwith a polymeric substance, such as ethylcellulose, hydroxypropylcellulose, ethylene/vinylacetate, polyvinyl pyrrolidone, and the like,to provide the composition in gel form, which may be dissolved insolvent, such as methylene chloride, evaporated to the desiredviscosity, and then applied to backing material to provide a patch.

The present invention also provides therapeutic compositions, comprisinga CP-d/n-ATF5 and, optionally, a pharmaceutically-acceptable carrier.The pharmaceutically-acceptable carrier must be “acceptable” in thesense of being compatible with the other ingredients of the composition,and not deleterious to the recipient thereof. Thepharmaceutically-acceptable carrier employed herein is selected fromvarious organic or inorganic materials that are used as materials forpharmaceutical formulations, and which may be incorporated as analgesicagents, buffers, binders, disintegrants, diluents, emulsifiers,excipients, extenders, glidants, solubilizers, stabilizers, suspendingagents, tonicity agents, vehicles, and viscosity-increasing agents. Ifnecessary, pharmaceutical additives, such as antioxidants, aromatics,colorants, flavor-improving agents, preservatives, and sweeteners, mayalso be added. Examples of acceptable pharmaceutical carriers includecarboxymethyl cellulose, crystalline cellulose, glycerin, gum arabic,lactose, magnesium stearate, methyl cellulose, powders, saline, sodiumalginate, sucrose, starch, talc, and water, among others.

The CP-d/n-ATF5 formulations of the present invention can be prepared bymethods well-known in the pharmaceutical arts. For example, theCP-d/n-ATF5 can be brought into association with a carrier or diluent,as a suspension or solution. Optionally, one or more accessoryingredients (e.g., buffers, flavoring agents, surface active agents, andthe like) also can be added. The choice of carrier will depend upon theroute of administration. The pharmaceutical composition would be usefulfor administering the CP-d/n-ATF5 to a subject to treat a tumor and/orneoplastic cell, as discussed herein. The CP-d/n-ATF5 is provided in anamount that is effective to treat the tumor and/or neoplastic cell in asubject to whom the pharmaceutical composition is administered. Thatamount may be readily determined by the skilled artisan, as describedabove.

Compositions of the present disclosure can further include othertherapeutic agents. For example, they can include any one or moreanti-cancer agents. In certain embodiments, the one or more anti-canceragent will be selected from the group consisting of: alkylating agents;anti-metabolites; anti-microtubule agents; topoisomerase inhibitors,antibiotics, and antibodies/antibody-drug conjugates. The amounts ofthose anti-cancer agents in compositions of the present disclosure can,in certain embodiments, be reduced as compared to normal doses of suchagents administered in a similar fashion.

The present invention also provides kits for use in treating and/orpreventing tumors and/or neoplastic cells. In certain embodiments thekits comprise a CP-d/n-ATF5 molecule and a pharmaceutically acceptablecarrier. In certain embodiments the kits further comprise a means foradministration, for example, but not limited to, a pre-filled syringe,pen, pump, or other pre-filled device for parenteral administration ofthe CP-d/n-ATF5. In certain embodiments, the kits will comprise aCP-d/n-ATF5 formulated for intranasal delivery and a means forintranasal administration, such as, but not limited to, a metered doseinhaler or other commercially available administration device which canbe used or can be adapted for nasal administration of a composition asdescribed herein.

The present invention is described in the following Examples, which areset forth to aid in the understanding of the invention, and should notbe construed to limit in any way the scope of the invention as definedin the claims which follow thereafter.

EXAMPLES CP-d/n-ATF5 Example Materials & Methods

Truncation of d/n ATF-5. Using pQC eGFP-d/n-ATF5 plasmid (see,Angelastro et al., J Neurosci 2003; 23(11):4590-600) as template, PCRusing upstream primer 5′-TCC GCG GCC GCA CCG GTC GCC-3′ (SEQ ID NO: 46)and downstream primer 5′-CTC GAG GAT ATC TCA GTT ATC TAC ACT GAC TCT GCCCTC TCC CTC AG-3′ (SEQ ID NO: 47) truncated 75 base pairs from the 3′plasmid. Electrophoretically purified eGFP-d/n ATF5-tr (tr=truncated)cDNA was ligated into pGEM-T Easy Cloning vector (Promega), transformedinto DH5α cells, and plated onto LB agar-Ampicillin plates withblue-white selection. Selected colonies were amplified overnight in LBplus ampicillin. Plasmids isolated from the culture (mini-prep,Invitrogen) were digested with AgeI and EcoRV followed by agarose gelelectrophoresis to verify d/n-ATF5-tr insertion and inserts underwentDNA sequencing for verification. AgeI/EcoRV digested eGFP-d/n-ATF5-trcDNA was ligated into AgeI/EcoRV-digested purified pQCXIX (Clontech)expression vector. The ligation mixture was used to transform DH5αbacteria and the product was verified by AgeI/EcoRV digestion and gelelectrophoresis from minipreps of bacterial cultures and DNA sequencingof uncut plasmid. The pQC-eGFP-d/n-ATF5-tr plasmid was grown in Maxiprep(Invitrogen).

CP-6XHis-Pen-Flag-tagged-d/n-ATF5 protein (“6XHis” disclosed as SEQ IDNO: 16) production and bioassay. To createCell-Penetrating-6XHis-Penetratin-Flag-tagged-d/n-ATF5-tr(CP-6XHis-Pen-Flag-tagged-d/n-ATF5-tr) cDNA (“6XHis” disclosed as SEQ IDNO: 16), PCR was first employed using upstream primer 5′-TTA ATT AAG CCGCCA TGG ATG CGT CAA ATT AAA ATT TGG TTT CAA AAT CGT CGT ATG AAA TGG AAAAAA ATG GAC TAC AAG GAC GAT GAT-3′ (SEQ ID NO: 48) and downstream primer5′-CTC GAG GGA TCC TCA GTT ATC TAC ACT GAC TCT GCC CTC TCC CTC AG-3′(SEQ ID NO: 49) and pQC-Flag-d/n-ATF5-tr as template. The product waspurified after gel electrophoresis and ligated into pGEM-T Easy cloningvector. This was transformed into DH5α cells and for white colonyselection. Miniprep clones were digested with EcoRV followed by gelelectrophoresis and sequencing of uncut plasmid to verify the insert. Toinsert a 6xHis tag (SEQ ID NO: 16) at the N-terminus, Pen-d/n-ATF5-RP-trwas cloned into the pET-15b expression vector (Novagen). Both pET-15band pGEMT-Pen-d/n-ATF5-RP-tr vectors were digested with Nde-1 and BamH1and the cut Pen-d/n-ATF5-RP-tr was separated from pGEMT by gelelectrophoresis. Likewise, cut pET-15b was separated from the insert bygel electrophoresis. Both pET-15b and Pen-d/n-ATF5-RP-tr were excisedfrom the gel and purified and then ligated using T4 DNA ligase. Theligated material was used to transform DH5α cells and colonies selected.Mini-prepped constructs were digested with Xba-1 and EcoRV to verify thepresence of vector and Pen-d/n-ATF5-RP insert using gel electrophoresis.The pET-15b-Pen-d/n-ATF5-RP was verified by DNA sequencing for correctorientation and sequence.

To generate CP-6xHis-Pen-Flag-tagged-d/n-ATF5 protein (“6xHis” disclosedas SEQ ID NO: 16), the expression construct was transformed into BL21DE3 pLysS cells (Novagen). Colonies were selected and amplified in LB.Peptide production was induced with 1 mM IPTG and verified by SDS-PAGE.Once protein induction was verified, extractions were accomplished withdetergent-based BugBuster master mix system (Novagen). Isolation andpurification of the Pen-Flag-tagged-d/n-ATF5 peptide was accomplishedusing its N-terminal 6xHIS-tag (SEQ ID NO: 16) and cobalt spin columnsystem (HisPur; Thermo Fisher). Purified peptide was desalted andbuffer-exchanged to PBS using Zeba de-salt spin columns (Thermo Fisher)or G-25 Sephadex (GE Health Care). Desalted protein was sterile-filteredusing 0.20 μm polyethersulfone membrane syringe filters (Sarstedt).Lastly, the peptide was concentrated to 1-2 mg/ml using Amicon Ultra-4centrifugal filter devices (3000 MW Cutoff).

A control peptide (Pen-Flag-tagged-Control) was created and producedusing the same methodology by eliminating the d/n-ATF5 portion of theconstruct using PCR upstream primer5′-CCCGGGCATATGCGTCAAATTAAAATTTGGTTT-3′ (SEQ ID NO: 50) and downstreamprimer 5′-CTCGAGGGATCCTCAGTTATCTAGTCTGGGTCTCTTCC-3′ (SEQ ID NO: 51).

Mass Spectroscopy. Linear MALDI-TOF analysis for nominal molecular massmeasurement: Matrix-assisted laser desorption/ionization (MALDI)measurements were acquired on a MALDI-TOF/TOF mass spectrometer (4700Proteomics Analyzer, AB Sciex) equipped with a 200 Hz ND-YAG lasersource (355 nm). Samples were spotted onto the MALDI plate with anequivolume of MALDI matrix (sinapinic acid in 50% ACN/0.1% FA, Fluka)and air dried. The instrument was operated at an accelerating voltage of20 kV. Spectra were taken from signal averaging of 4,000 laser shots.Mass Spectra analyses were performed in positive ion linear mode with amass range of 10,000-60,000 m/z. Data were further analyzed by DataExplorer 4.5 (AB Sciex).

LC-MS analysis: Samples were injected onto an Aeris Widepore XB-C8column (3.6μ, 2.10×50 mm). A standard reverse phase gradient was runover 8 minutes at flow rate of 250 μl/min and the eluent monitored by aLTQ-OrbitrapXL mass spectrometer (Thermo Fisher) in profile mode. IonMax Source (Thermo Fisher) was used as the electrospray ionizationsource and source parameters were 5 kV spray voltage, capillarytemperature of 275° C. and sheath gas setting of 20. Spectral data wereacquired at a resolution setting of 15,000 FWHM with the lockmassfeature.

Bioactivity of the pQC-eGFP-d/n ATF-5tr product (C-terminally truncatedd/n-ATF5). Purified pQC-eGFP-d/n-ATF5-tr, full-length pQC-eGFP-d/n-ATF5positive control or pQC-eGFP negative control plasmids were transfectedinto rat C6 glial cells in 24 well plates using Lipofectamine 2000(Invitrogen). After 48 hours, cells were stained with DAPI and 10 randomfields were viewed under fluorescent microscopy at 40×. Cells displayingfragmented, condensed chromatin were scored as apoptotic and quantifiedrelative to total cells (n=3 independent experiments).

Cell penetrating (CP)-6xHis-Pen-Flag-tagged-d/n-ATF5 (“6xHis” disclosedas SEQ ID NO: 16) bioassay. For peptide bioassays, rat C6 glioblastomacells were maintained in serum-free DMEM for 2 hours, and then inDMEM/0.5% FBS without or with 3 μM Penetratin (Pen)-d/n-ATF5-RP peptideor (Penetratin) Pen-control-RP. After 5 days, cells were stained withDAPI and percent of apoptotic cells determined as described above.

Imaging of internalized Pen-d/n-ATF5-RP (Recombinant Protein). Rat C6cells (from Jeff Bruce; Columbia University, New York; authenticated2004 by grafting into Rat brain Angelastro et al., Oncogene 2006;25(6):907-16) and U87 cells (purchased and authenticated from the ATCC)were plated on fibronectin-coated confocal microscopy coverslips andmaintained overnight. 3 μM each of Pen-d/n-ATF5-RP or Pen-Control-RPwere added to wells and incubated for 1, 2, 4, or 24 hours. Cells werewashed 3× with PBS to remove extracellular peptide and stained withprimary mouse anti-FLAG antibody (Sigma-Aldrich) overnight followed byincubation for two hours with secondary anti-mouse Alexa-568(Invitrogen). Microscopy used a Carl Zeiss Axiovert 200 with Axiocamvideo capture or Delta Vision Deconvolution microscope at 0.1-μm opticalsections enhanced by Huygens Deconvolution Software. Images of xy and yzplanes confirmed co-localization of Pen-d/n-ATF5-RP and DAPI staining.

Retrovirus-induced mouse glioblastoma model and treatment withPen-d/n-ATF5-RP. As described previously (Arias et al., Oncogene 2012;31(6):739-51), adult mice were anesthetized and underwent stereotaxicinjection of retrovirus expressing PDGF-B and p53-shRNA to generatemalignant gliomas. Analgesics were given immediately after surgery.Injected mice were monitored post-surgically and throughout the studyperiod, which ranged from 52 to 438 days. Pen-d/n-ATF5-RP orPen-Control-RP was administered to tumor-bearing animals in treatmentsof four subcutaneous or intraperitoneal injections, spaced 1-2 hoursapart. The doses were 1 mg/kg (200 μl, 0.9% saline) for each injection.In some experiments as indicated, dosing was repeated 5 days later.Animals injected with 0.9% saline at the same dosing schedule and volumeserved as controls.

Brain sectioning and staining. As previously described, (Arias et al.,Oncogene 2012; 31(6):739-51), mice were euthanized by deep isofluraneanesthesia followed by trans-cardial perfusion with 10% formalin. Brainswere fixed in 4% paraformaldehyde, incubated overnight in 30% sucroseand were mounted in OCT medium, frozen and cut into 14-μm coronalsections. In other cases as indicated, brains of perfused mice wereincubated in 10% formalin/PBS for 4-7 days and then paraffin-embedded.Paraffin sections were subjected to antigen retrieval as described(Schrot et al., J Neurooncol 2007; 85(2):149-57). Sections were stainedwith DAPI and the following: Anti-Flag M2 (1:200; Sigma-Aldrich), rabbitanti-Flag (1:1000, Cell Signaling), rabbit anti-HA (4 μg/ml; sc-805Santa Cruz Biotechnology), or TUNEL (Roche) and Anti-Flag M2. Sectionswere visualized with a DAPI filter and immunofluorescence (Alexa488/568; Invitrogen) or colorimetrically with diaminobenzidine or fastred (Mach2; Biocare Medical) and photographed on a Carl Zeiss Axiovert200 with Axiocam video.

MRI analysis. Anesthetized (isoflurane and oxygen) mice were fittedintravenously with a 30 gauge catheter, and positioned head first, proneon the scanner bed. MRI acquisitions were performed on a Bruker Biospec7 Tesla magnet operating Paravision v5.1 and outfitted with a 116-mmdiameter gradient with integrated shim control. Maximum gradientstrength was 450 mT/m. A cross coil configuration was used for imagingbrains and a 72-mm ID linear coil was used for RF transmission and a 4channel phased array coil for RF reception. Pre-contrast and 1 minutepost contrast images were acquired with FLASH_3Dslab. Gadolinium wasinjected intravenously at a dose of 1 μl/g body weight.

Results

Generation of a cell-penetrating form of d/n-ATF5. A modifiedcell-penetrating form of d/n-ATF5 that could be delivered systemicallywas prepared as outlined herein. This has a potential advantage of rapidbiodistribution, reduced immune response, passage through the bloodbrain barrier, entry into cells, and the capacity reaching widelydispersed tumor cells. The original d/n-ATF5 is an N-terminallytruncated form of ATF5 that includes the wild-type leucine zipper domainwith an amphipathic α-helical sequence with leucine repeats at everyseventh residue replacing the DNA binding domain [Angelastro et al., JNeurosci 2003; 23(10:4590-600]. The resulting protein is capable ofinteracting with ATF5 and its binding partners via the enhanced leucinezipper region, but not with DNA, and consequently acts as an effectived/n suppressor of ATF5 actions [Angelastro et al., J Neurosci 2003;23(11):4590-600; Vinson et al., Genes Dev 1993; 7(6):1047-58]. Deletionof the N-terminal domain substantially stabilizes d/n-ATF5 againstdegradation [Lee et al., Developmental Neurobiology 2012; 72(6):789-804;Uekusa et al., Biochem Biophys Res Commun 2009; 380(3):673-8]. To designa deliverable form of d/n-ATF5, the last 25 amino acids of the proteinwere first truncated, which includes the C-terminal two valine/valineheptad repeats. Transfection of this deleted construct into C6glioblastoma cells showed equal effectiveness as the full lengthd/n-ATF5 in promoting apoptosis (FIG. 1).

To generate a cell-penetrating form of the C-terminally truncatedd/n-ATF5 (d/n-ATF5-tr), an N-terminally Flag-tagged d/n-ATF5-trconstruct N-terminally fused to a 6x histidine repeat (SEQ ID NO: 16)followed by a penetratin sequence was designed (FIG. 2A). Penetratinsequence is a 16-amino acid motif from the Antennapedia homeodomainprotein permitting passage of fused cargos through biological membranesinto cells [Dupont et al., Methods in molecular biology 2011;683:21-9.]. Milligram quantities of the protein (designatedPen-d/n-ATF5-Recombinant Protein (RP)) were generated by expression inbacteria followed by purification by cobalt resin affinitychromatography using the 6xHis sequence (SEQ ID NO: 16). SDS-PAGE showedthe purified preparations were more than 95% homogeneous with minorspecies including what appeared to be aggregated protein multimers.Calculated Mr of Pen-d/n-ATF5-RP with normal bacterial removal of theN-formylmethionine is 12,949.18 Da, but the major purified product showsan apparent molecular mass between 25-28 KDa by SDS-PAGE (FIG. 2A). Wildtype ATF5 and the ATF5 leucine zipper can migrate anomalously whensubjected to SDS-PAGE and so high resolution LC-HRMS was employed toverify the correct molecular weight of Pen-d/n-ATF5-RP as well as itssolution state. The deconvoluted spectra revealed the most abundant formto be the predicted 12,948.7 Da monomer, with a low amount of dimer at25,897.5 Da (FIG. 2B). Prior studies have also shown that recombinantwild type full-length ATF5 or the bzip domain of ATF5 can form dimers invitro. Finally, as a control for Pen-d/n-ATF5-RP, a peptide(Pen-Control-RP) was generated by similar means that lacks thed/n-ATF5-tr sequence (FIG. 2A). The purified recombinant control (with acalculated molecular mass of 7,099.98 Da) migrated at an apparent MW of7,100 Da by SDS-PAGE (FIG. 2A).

Because Pen-d/n-ATF5-RP is designed for systemic administration,stability in presence of human serum at 37° C. was shown with nosignificant degradation at 8 h and a mean loss of 28% of full-lengthprotein by 48 h (FIG. 2C).

Pen-d/n-ATF5-RP rapidly enters and causes apoptosis of culturedglioblastoma cells. Before carrying out animal experiments, the abilityof Pen-d/n-ATF5-RP to enter and kill glioblastoma cells in culture wasverified. When added to serum-containing cultures of rat C6 and humanU87 glioblastoma cells, both Pen-control-RP and Pen-d/n-ATF5-RP werereadily detectable in the cells within 2-4 h and remained detectable forat least 24 h (FIG. 3A, 3B). Confocal microscopy revealed that thepeptides were present in both the cytoplasmic and nuclear compartments(FIG. 3A).

C6 cultures exposed to Pen-control-RP and Pen-d/n-ATF5-RP were alsoassessed for apoptotic cell death. The Pen-Control-RP treated culturesshowed background levels of apoptotic death similar to that innon-treated cultures, whereas cultures treated with Pen-d/n-ATF5-RPshowed greatly increased numbers of dying cells (FIG. 4). These actionsare similar to what has been previously reported for multiple rodent andhuman glioblastoma cells transfected with d/n-ATF5 constructs or exposedto ATF5 siRNA [Angelastro et al., Oncogene 2006; 25(6):907-16; Arias etal., Oncogene 2012; 31(6):739-51; Sheng et al., Nat Med 2010;16(6):671-7; Dluzen et al., The Journal of biological chemistry 2011;286(9):7705-13].

Systemically-delivered Pen-d/n-ATF5-RP crosses the blood brain barrier,enters cells and selectively triggers rapid, selective apoptotic deathof glioma cells. To test the capacity of Pen-d/n-ATF5-RP to reach andtreat primary brain tumors, a model in which gliomas are generated bystereotactic injection of PDGF-B-HA/shRNA-p53 retrovirus into the adultmouse brain was used. The tumors are presumably derived from endogenousdividing progenitor cells and closely resemble infiltrative humangliomas ranging from stages II-IV. The tumors were detectable as earlyas 52 days post-injection by MRI (see below) and were histologicallyidentifiable by the presence of the HA tag as well as by highcellularity, hyperchromatic nuclei, and elevated Ki67 staining.

In an initial set of experiments, Pen-d/n-ATF5-RP, saline orPen-Control-RP was delivered intraperitoneally to tumor-bearing mice ina single set of four injections each of 1 mg/kg at intervals of 1-2 h.The mice were sacrificed 16-64 hours after the last injection and thefixed brains were stained with anti-Flag antibody to detectPen-d/n-ATF5-RP or with anti-HA to mark PDGF-B-HA expressing tumorcells, and for TUNEL to identify dying cells. At 16 h, both tumor andnormal brain cells (in the contralateral hemisphere from the tumor)showed Flag staining indicative of extensive uptake of Pen-d/n-ATF5-RP;there was no signal with saline injection (FIG. 5A-5C). Flag stainingwas still evident at 40 h after treatment and was detectable, though atreduced levels at 64 h (FIG. 6A-6D). While normal brain tissue showed noTUNEL staining (FIG. 5B), there was extensive TUNEL staining within thetumors one day after treatment with Pen-d/n-ATF5-RP (FIG. 5A). Little orno TUNEL signal was observed in tumors of animals treated with saline(FIG. 5C). Co-localized TUNEL and PDGF-B-HA+ tumor marker stainingcontinued to be evident at 64 h after Pen-d/n-ATF5-RP treatment, but thesignals indicated cell degeneration and fragmentation (FIG. 5E) comparedwith cells treated with this peptide for 16 h (FIG. 5A) or withPen-Control-RP peptide (FIG. 5D).

To enhance the potential long-term therapeutic efficacy ofPen-d/n-ATF5-RP administration, a treatment protocol was devised inwhich tumor-bearing animals received two sets of subcutaneousinjections, 5 days apart, each as described above. Tumors of miceassessed two days after the second treatment (7 days after initialtreatment) showed patterns of HA and TUNEL staining, that, like 64 hafter a single set of treatments, indicated cell degeneration andfragmentation (FIG. 5F).

Full body necropsy of non-tumor bearing animals one (n=2) or two days(n=2) after completion of the above dual treatment regimen revealed noevident pathological lesions to internal organs and no evidentabnormalities of the cerebrum or cerebellum (FIG. 7A-7D′ and Table 1).In addition, a liver-kidney serum chemistry panel carried out 1 dayafter the second set of Pen-d/n-ATF5-RP injections indicated no damageto either organ (Table 1; n=2).

TABLE 1 Results from gross necropsy of organs, H&E staining of tissuesections and liver-kidney function blood panel of mice treated withPen-d/n-ATF5-RP. 1 day 1 day 2 day 2 day 6 Month 6 Month after afterafter after Post-tumor Post-tumor Gross Pen-d/n- Pen-d/n- Pen-d/n-Pen-d/n- treatment treatment Necropsy/ ATF5-RP ATF5-RP ATF5-RP ATF5-RPwith with H&E treatment treatment treatment treatment Pen-d/n- Pen-d/n-slides #1 #2 Control #1* #2* ATF5-RP #1 ATF5-RP #2 Cerebrum, No gross Nogross No gross No gross No gross No gross No gross cerebellum, nasallesion/No lesion/No lesion/No lesion/No lesion/No lesion/No lesion/Nocavity, liver, significant significant significant significantsignificant significant significant kidneys, spleen, changes of changesof changes of changes of changes of changes of changes of pancreas,heart, pathological pathological pathological pathological pathologicalpathological pathological lungs, trachea, significance significancesignificance significance significance significance significanceesophagus, thymus, salivary glands, GI tract, hind limb muscles, urinarybladder, reproductive tract Liver-kidney function panel Alkaline AlanineAspartate Blood Total Albu- Phospha- Transam- Transam- Urea Creat- Bili-Total min tase inase inase Nitrogen inine rubin Protein Lipe- Hemo-Mouse g/dL U/L U/L U/L mg/dL mg/dL mg/dL g/dL mia lysis Male, one 3.9552.9 55.4 128.7 20.4 0.072 0.089 5.87 None None day after Pen-d/n-ATF5-RP treatment #1 Male, one 3.35 46.0 21.6 66.6 19.7 0.061 0.115 5.21None None day after Pen-d/n- ATF5-RP treatment #2 JAX 3.77 ± 0.247 78.3± 32.6 52.7 ± 19.6 152 ± 92.6 23.7 ± 3.47 0.167 ± 0.258 0.695 ± 0.1676.10 ± 0.396 Not Not database Listed Listed strain range (males) Theindicated organs were collected from mice sacrificed at 1 day, 2 daysand >6 months (190 days and 183 days, corresponding to mice witheradicated tumors in FIG. 3A-3B, and FIG. 8A-8F and FIG. 11A-11E,) afterthe second of two sets of subcutaneous treatments with Pen-d/n-ATF5-RPas described in the text. The >6 month animals had MRI-detected tumorsbefore treatment and no histologically detectable tumors at the time ofsacrifice. All other animals were not tumor-bearing. The control mousewas untreated. The organs were evaluated for gross pathological changesand then fixed, paraffin embedded and used for preparation of slide-mounted 5 μm sections. The slides were stained with H&E and examinedmicroscopically for possible pathological changes. Gross pathologicalanalysis and evaluation of sections were carried out by the ComparativePathology Laboratory at the UC Davis School of Veterinary Medicine.*Regional coagulative necrosis in the liver and focal linear pneumoniaof the lung were observed due to inadvertent needle penetration duringthe injections. For liver-kidney function panel, blood samples wereobtained 1 day after the second of two sets of subcutaneous treatmentswith Pen- d/n-ATF5-RP as described in the text. The animals were nottumor-bearing. The analysis was carried out by the Comparative PathologyLaboratory at the UC Davis School of Veterinary Medicine. The data forthe strain (C57BL/6J) range was obtained from the Mouse Phenome Databaseat the Jackson Laboratory(http://phenome.jax.org/db/q?rtn=meas/catlister&req=Dblood--clinical%20chemistryqqq44&reqstrainid=7).

Systemically delivered Pen-d/n-ATF5-RP promotes rapid regression ofmouse gliomas without recurrence as indicated by MRI and histology.Whether systemic administration of Pen-d/n-ATF5-RP promoted prolongedregression of gliomas in a mouse model was assessed. To achieve this MRI(post-contrast enhanced 3D FLASH T1 weighted) was used to assess tumorsbefore and at various times after treatment with Pen-d/n-ATF5-RP,Pen-control-RP or no treatment. In many cases, the tumors were eithermultifocal or present in both hemispheres prior to treatment (FIG.8A-8F, FIG. 9A-9E, FIG. 10, and FIG. 11A-11E). The peptides wereinjected subcutaneously using the two treatment protocol describedabove. Treatments commenced only after the presence of tumors wasverified by MRI and were randomly assigned.

As anticipated, in no case was tumor regression observed as assessed byMRI in untreated animals (n=5) or animals treated with Pen-control-RP(n=4). A typical example for an animal treated with control peptide isshown in FIG. 8A-8F. Tumor presence was verified by histology on brainsof animals those either died or were sacrificed after exhibitingmoribund behavior or that survived beyond the study endpoint (6 monthsafter MRI tumor detection). The tumors were HA+ (FIG. 8E and FIG. 10),indicating the presence of the tagged PDGF-B and exhibitedhyperchromatic nuclei (FIG. 8D) and elevated Ki67 staining typical ofgliomas (FIG. 8F). The infiltrative tumor boundaries matched those inthe MRI images (FIG. 8A-8F).

For mice treated with Pen-d/n-ATF5-RP, MRI revealed significantreduction (2/5; FIG. 9A-9E and FIG. 11A-11E) or un-detectability (3/5)of tumor signals at 8 days after treatment (the earliest time monitored)and full loss of detectable tumor signal within 3 weeks (n=7/7). Whenassessed by MRI at 176-225 days after peptide treatment, 7/7 miceassessed were tumor-free (see for example, FIG. 8A-8F, FIG. 11A-11E andFIG. 12B). Thus, Pen-d/n-ATF5-RP treatment appeared to rapidly cleargliomas without MRI-detectable recurrence for at least 6-7 months.

Postmortem histology (n=6; 183-259 days after treatment; 190-305 daysafter tumor detection) corroborated the MRI findings of tumorregression/eradication (FIG. 8A-8F, FIG. 11A-11E, and FIG. 12C). As inthe rest of the brain, areas that initially had been tumor positive byMRI, showed an absence of hyperchromatic nuclei or high cellularity orelevated Ki67 staining (FIG. 8A-8F and FIG. 11A-11E). There was also nostaining (other than scarce scattered single cells) for PDGF-B-HA+ (FIG.8A-8F and FIG. 11A-11E). There were however, foci of GFAP+ cells,suggesting glial activation and scarring in the areas where tumors hadbeen present (FIG. 8A-8F and FIG. 11A-11E).

Systemically delivered Pen-d/n-ATF5-RP promotes long-term survival whilemaintaining normal brain and tissue integrity. All eight tumor-bearingmice treated with Pen-d/n-ATF5-RP survived to the nominal 180 dayendpoint of the study after detection of tumors (FIG. 12A). In contrast,6/9 control mice died within this time. In a past study 40% (n=16) ofmice died within 180 days of tumor initiation [Arias et al., Oncogene2012; 31(6):739-51].

In addition to the 6 mice that were sacrificed for histology at 6-8months after Pen-d/n-ATF5-RP treatment, 2 animals have been maintainedfor a 12-month post-treatment time.

Other than the absence of tumors and the presence of glial scarring inareas of prior tumor localization, H&E staining of the brains of theanimals sacrificed 6-8 months after Pen-d/n-ATF5-RP treatment indicatedno evident abnormalities and both the subventricular and hippocampalsubgranular zones appeared normal (FIG. 7A-7D′). Additionally, theweights of the treated mice prior to sacrifice were either within (4/6)or greater than (2/6) one standard deviation of the mean weight ofage-matched controls given in the Mouse Phenome Database at the JacksonLaboratory (phenome.jax.org/db/q?rtn=strains/details&strainid=7). Twomice were also subjected to full body necropsy at >6 months of treatment(190 days and 183 days, corresponding to mice with eradicated tumors inFIG. 8A-8F and FIG. 11A-11E, respectively). No pathological changes wereseen in any of the organs surveyed (Table 1).

Discussion

The findings presented herein show that Pen-d/n-ATF5-RP enters andpromotes apoptotic activity in cultured GBM cells and that whensystemically administered to animals, crosses the blood brain barrier,enters brain and tumor cells and causes massive tumor cell death andlong-term tumor regression/eradication without apparent harm to normaltissues.

Another feature of the study presented herein was that the treatedtumor-bearing animals survived for at least 6-12 months. By contrast,2/3 of control animals died or showed morbidity within 189 days of tumordetection and all were tumor positive at death or at the 6 month point.Taken together, the results presented herein provide proof that a cellpenetrating form of d/n-ATF5 can be used to treat malignant gliomas.

A model in which malignant gliomas were induced in adult mice byretrovirally expressed PDGF-B and p53 shRNA, presumably bytransformation of PDGF-α-receptor+ neural progenitors andoligodendrocyte precursors, was used in the instant study. Such tumorsresemble high grade human glioma [Arias et al., Oncogene 2012;31(6):739-51] and, like the latter, are highly diffuse and relativelylarge and can invade both hemispheres. Given the wide expression of ATF5in human GBMs and lower grade gliomas and the variety of human androdent-derived GBM cell lines (with and without compromised p53 andPTEN) that express and require ATF5 for survival [Arias et al., Oncogene2012; 31(6):739-51], it is expected that, based on the data presentedherein, a range of malignant glioma cell types will be susceptible totreatment with cell-penetrating d/n-ATF5. Furthermore, althoughmalignant gliomas are the focus of this study, it is significant to notethat ATF5 is expressed by a wide variety of carcinomas [Sheng et al.,Oncotarget 2010; 1(6):457-60; Chen A et al., International journal ofgynecological pathology 2012; 31(6):532-7; Fernandez et al., Oncogene2004; 23(29):5084-91; Kong et al., Experimental and therapeutic medicine2011; 2(5):827-831; Monaco et al., Int J Cancer 2007; 120(9):1883-90;and Hu et al., Anticancer research 2012; 32(10):4385-94], and thatculture studies have shown apoptotic actions of d/n-ATF5 or ATF5 siRNAon tumor cells from a diverse range of tissues. [Sheng et al.,Oncotarget 2010; 1(6):457-60; Chen A et al., International journal ofgynecological pathology 2012; 31(6):532-7; Monaco et al., Int J Cancer2007; 120(9):1883-90; and Hu et al., Anticancer research 2012;32(10):4385-94]. Thus, based on the data presented herein, a diverserange of cancers will be susceptible to treatment with cell-penetratingd/n-ATF5.

An important aspect of the instant study was that althoughPen-d/n-ATF5-RP promoted regression/eradication of tumors, it had noapparent adverse effects on normal tissue. It is significant thattreated animals survived without apparent effect for at least 6-12months and that no evident acute or long term tissue damage wasobserved. In addition, any potential negative effects of Pen-d/n-ATF5-RPmay be mitigated by the limited duration of treatment.

Addition Cell Lines and CP-d/n-ATF5 Compositions TAT-d/n-ATF5 PromotesApoptotic Death of Cultured Melanoma MEL501 Cells

TAT-linked dominant-negative ATF5 peptide was added to medium of MEL501melanoma cells at the concentrations (in μM) indicated in FIG. 13. Fourdays later the cells were stained with Hoescht dye and the cells werestained for proportion with apoptotic nuclei. As illustrated in FIG. 10,TAT-d/n/ATF5 promoted apoptosis in a dose-dependent fashion.

TAT-d/n-ATF5 Reduces Expression of Endogenous ATF5 in Cultured U373Glioblastoma Cells

TAT-linked dominant-negative ATF5 peptide was added to medium of U373glioblastoma cells at the concentrations (in μM) indicated in FIG. 14for 17 hrs day and the cells were then harvested and analyzed by Westernimmunoblotting for levels of endogenous ATF5. Note that the TAT-d/n-ATF5greatly reduces expression of endogenous ATF5. As previous studies haveshown that tumor cells require endogenous ATF5 to survive, but withoutbeing bound by theory, the mechanism of action by which thecell-penetrating TAT-ZIP peptide kills may be by causing loss of theendogenous ATF5 protein. Note also the smear above the endogenous ATF5when the TAT-ZIP peptide is present. This suggests that TAT-ZIP reducesendogenous ATF5 by causing its ubiquitination and proteasomaldegradation.

TAT-d/n-ATF5 induces Expression of the Pro-Death Gene DDIT3

TAT-d/n-ATF5 (TAT-ZIP) peptide induces expression of the pro-death geneDDIT3 (CHOP) in various tumor cell lines. Cells were treated withTAT-d/n-ATF5 for the times and doses (in μM) indicated in FIG. 15 andthen harvested and analyzed by Western immunoblotting for expression ofCHOP and other non-responsive proteins. Note the elevation of CHOP inall cases. Since CHOP may promote cell death, these data indicate thatinduction of CHOP protein may be one mechanism by which TAT-d/n-ATF5kills tumor cells.

Silencing of CHOP Protein with siRNA Partially Protects U87 Cells fromTAT-d/n-ATF5

Silencing of CHOP protein with siRNA (top Western immunoblot of FIG. 16)partially protects U87 cells from death caused by TAT-d/n-ATF5 peptide.Cells were treated with siCHOP to silence CHOP expression (top Westernimmunoblot of FIG. 16) or with control siRNA. They were then exposed toTAT-d/n-ATF5 for 2 days and assessed for proportion of cells withapoptotic nuclei. The data indicate that part of the mechanism by whichTAT-d/n-ATF5 kills tumor cells is by increasing their expression of CHOPwhich in turn mediates death.

TAT-D/N-ATF5 Down-Regulates BCL2 Survival Protein

TAT-D/N-ATF5 down-regulates BCL2 survival protein. As outlined in FIG.17, cultured U87 human glioblastoma cells were treated with theindicated concentrations of TATZIP (TAT-d/n-ATF5 peptide) (in μM) for 30hrs. The cells were then harvested and assessed by Westernimmunoblotting for expression of the survival protein BCL2. Thesefindings indicate that in addition to elevating pro-death CHOP,TAT-d/n-ATF5 may also kill tumor cells by reducing their levels of theBCL2 survival protein.

TAT-D/N-ATF5 Synergizes with Temozolomide to Kill Cultured U87Glioblastoma Cells

TAT-D/N-ATF5 synergizes with temozolomide (TMZ) to kill cultured U87glioblastoma cells. As outlined in FIG. 18, cells were cultured for oneday with sub-lethal levels of TAT-d/n-ATF5 (TZIP 1 μM) and TMZ (50 μM)either separately or in combination, and then assessed for proportion ofcells with apoptotic nuclei. TMZ is presently the first-line treatmentfor human GBM. The data reveal that TAT-d/n-ATF5 not only functions inpresence of TMZ, but that the two drugs act in synergy to kill GBMcells. This indicates that TAT-d/n-ATF5 can be administered to patientswho are taking TMZ.

TAT-D/N-ATF5 Decreases Viability of U87, U373, and MSG Cells

As outlined in FIG. 19, recombinant TAT-d/n-ATF5 (3 μM) treatment for3-5 days decreases viability of two human and one mouse GMB cell line asdetected using an MTA assay.

Synthetic PEN-D/N-ATF5 Decreases Viability of U87 Cells

As outlined in FIG. 20, synthetic PEN-d/n-ATF5 decreases cell viabilityof cultured U87 human glioblastoma cells. 5 days treatment at indicatedconcentrations (μM), as detected using an MTA assay.

TAT-D/N-ATF5 Promotes Cell Death of U87 Cells

As outlined in FIG. 21, Recombinant TAT-d/n-ATF5 promotes death ofcultured U87 human glioblastoma cells as indicated by Annexin V/PIstaining and flow cytometry. Proportions of viable cells are shown inlower left quadrant (88% control vs 58% treated). Dying cell proportionsare in the lower right and upper right quadrants (9% in controls vs 36%in treated).

Synthetic PEN-D/N-ATF5 Promotes Apoptosis of GS9-6 Cells

As outlined in FIG. 22, Synthetic PEN-d/n-ATF5 promotes apoptotic deathof primary GS9-6 human glioblastoma stem cells growing in culture asspheres. Data reflects 6 days of treatment. Data determined by AnnexinV/PI staining and flow cytometry.

Recombinant PEN-D/N-ATF5 Promotes Apoptosis of GS9-6 Cells

As outlined in FIG. 23, Recombinant PEN-d/n-ATF5 promotes apoptoticdeath of primary GS9-6 human glioblastoma stem cells growing in cultureas spheres. 5 days treatment. Data determined by Annexin V/PI stainingand flow cytometry.

Various publications are cited herein, the contents of which are herebyincorporated by reference in their entireties.

1-8. (canceled)
 9. A composition comprising a cell-penetratingdominant-negative ATF5 molecule consisting essentially of the sequenceRQIKIWFQNRRMKWKKLEQRAEELARENEELLEKEAEELEQENAELEGECQGLE ARNRELRERAESV(SEQ ID NO: 52).
 10. The composition of claim 9, wherein the compositionis a pharmaceutical composition.
 11. The composition of claim 9, whereinthe composition consists of a cell-penetrating dominant-negative ATF5molecule consisting essentially of the sequenceRQIKIWFQNRRMKWKKLEQRAEELARENEELLEKEAEELEQENAELEGECQGLE ARNRELRERAESV(SEQ ID NO: 52).
 12. A kit comprising a cell-penetratingdominant-negative ATF5 molecule consisting essentially of the sequenceRQIKIWFQNRRMKWKKLEQRAEELARENEELLEKEAEELEQENAELEGECQGLE ARNRELRERAESV(SEQ ID NO: 52).
 13. The kit of claim 12, wherein the cell-penetratingdominant-negative ATF5 molecule is in a pharmaceutically acceptablecarrier.
 14. A nucleic acid molecule encoding a cell-penetratingdominant-negative ATF5 molecule consisting essentially of the sequenceRQIKIWFQNRRMKWKKLEQRAEELARENEELLEKEAEELEQENAELEGECQGLE ARNRELRERAESV(SEQ ID NO: 52).
 15. A method of treating a neural tumor in a subject,the method comprising administering to the subject an effective amountof a cell-penetrating dominant-negative ATF5 molecule consistingessentially of the sequenceRQIKIWFQNRRMKWKKLEQRAEELARENEELLEKEAEELEQENAELEGECQGLE ARNRELRERAESV(SEQ ID NO: 52).
 16. The method of claim 15, wherein the neural tumor isa glioma.
 17. The method of claim 15, wherein the neural tumor is aglioblastoma.
 18. The method of claim 15, wherein the neural tumor is aneuroblastoma.
 19. The method of claim 15, further comprisingadministering to the subject temozolomide.
 20. The method of claim 15,wherein administration is oral, parenteral, intranasal, or transdermal.21. The method of claim 20, wherein the parenteral administration isintracranial, intrathecal, intramuscular, intraperitoneal, intravenous,or subcutaneous.